Jesse Donald Knotts was born in Morgantown, West Virginia, the youngest of four sons born to farmer William Jesse Knotts and his wife, Elsie Luzetta Knotts (nee Moore). His parents were married in Spraggs, Pennsylvania. His English paternal ancestors immigrated to America in the 17th century, originally settling in Queen Anne's County, Maryland. Knotts' brothers were named Willis, William, and Ralph.

Before he entered high school, Knotts began performing as a ventriloquist and comedian at various church and school functions. After high school, he traveled to New York City to try to make his way as a comedian, but returned home to attend West Virginia University when his career failed to take off. After his college freshman year, Knotts joined the United States Army and spent most of his service entertaining troops. He toured the Pacific Islands as a comedian as part of a G.I. variety show called Stars and Gripes. His ventriloquist act included a dummy named Danny "Hooch" Matador. In a TV Guide interview in the 1970s, he said that he had grown tired of playing straight man for a hunk of wood when he was in the Army. According to Knotts, he tossed the dummy overboard off a ship in the South Pacific. He swore that he could hear the dummy calling for help as the ship sailed on, leaving him bobbing helplessly in the waves. Knotts served in the United States Army from June 21, 1943 to January 6, 1946. Discharged in the rank of Technician Grade 5, which was the equivalent of a Corporal. During his military service, Knotts was awarded the World War II Victory Medal, Philippine Liberation Medal, Asiatic-Pacific Campaign Medal (with 4 bronze service stars), Army Good Conduct Medal, Marksman Badge (with a Carbine BAR) and Honorable Service Lapel Pin. Knotts returned to West Virginia University after being demobilized and graduated in 1948. He married and moved back to New York, where connections he had made while in the Special Services Branch helped him break into show business. In addition to doing stand-up comedy at clubs, he appeared on the radio, eventually playing the wisecracking, know-it-all character "Windy Wales" on a radio western called "Bobby Benson and the B-Bar-B Riders". Knotts got his first major break on television in the soap opera Search for Tomorrow where he appeared from 1953 to 1955. He came to fame in 1956 on Steve Allen's variety show, as part of Allen's repertory company, most notably in Allen's mock "Man in the Street" interviews, always as an extremely nervous man. He remained with the Allen program through the 1959-1960 season. From October 20, 1955 through September 14, 1957, Knotts appeared in the Broadway version of No Time for Sergeants, in which he played two roles, listed on the playbill as a Corporal Manual Dexterity and a Preacher. In 1958, Knotts appeared for the first time on film with Andy Griffith in the film version of No Time for Sergeants. In that film, Knotts reprises his Broadway role and plays a high-strung Air Force test administrator whose routine is disrupted by the hijinks of a provincial new recruit. In 1960, Andy Griffith was offered the opportunity to headline his own sitcom, The Andy Griffith Show (1960-1968). Knotts took the role of Barney Fife, the deputy--and originally cousin--of Sheriff Andy Taylor (portrayed by Griffith). Knotts's portrayal of the deputy on the popular show earned him five Emmy Awards for Best Supporting Actor in a Television Comedy, three awards for the first five seasons that he played the character. A summary of the show from the website of the Museum of Broadcast Communications describes Deputy Barney Fife: Self-important, romantic, and nearly always wrong, Barney dreamed of the day he could use the one bullet Andy had issued to him, though he did fire his gun on a few occasions. He always fired his pistol accidentally while still in his holster or in the ceiling of the court house, at which point he would sadly hand his pistol to Andy. This is why Barney kept his one very shiny bullet in his shirt pocket. In episode #196, Andy gave Barney more bullets so that he would have a loaded gun to go after a bad guy that Barney unintentionally helped escape. While Barney was forever frustrated that Mayberry was too small for the delusional ideas he had of himself, viewers got the sense that he couldn't have survived anywhere else. Don Knotts played the comic and pathetic sides of the character with equal aplomb and he received three Emmy Awards during the show's first five seasons. When the show first aired, Griffith was intended to be the comedic lead with Knotts as his straight man, similar to their roles in No Time for Sergeants. However, it was quickly discovered that the show was funnier with the roles reversed. As Griffith maintained in several interviews, "By the second episode, I knew that Don should be funny, and I should play straight." Knotts believed remarks by Griffith that The Andy Griffith Show would end after five seasons, and he began to look for other work, signing a five-film contract with Universal Studios. He was caught off guard when Griffith announced that he would continue the show after all, but Knotts's hands were tied. In his autobiography, Knotts admitted that he had not yet signed a contract when Griffith announced his decision; but he had made up his mind to move on, believing he would not get the chance again. Knotts left the series in 1965. His character's absence on the show was explained by Deputy Fife's having finally made the "big time," joining the Raleigh, North Carolina police force. Knotts went on to star in a series of film comedies that drew on his high-strung persona from the television series: he had a cameo appearance in It's a Mad, Mad, Mad, Mad World (1963), and starred in The Incredible Mr. Limpet (1964), The Ghost and Mr. Chicken (1966), The Reluctant Astronaut (1967), The Shakiest Gun in the West (1968), The Love God? (1969) and How to Frame a Figg (1971). Knotts reprised his role as Barney Fife several times in the 1960s: he made five guest appearances on The Andy Griffith Show (gaining him another two Emmy Awards), and he later appeared once on the spin-off Mayberry RFD, where he was present as best man for the marriage of Andy Taylor and his longtime love, Helen Crump. After making How to Frame a Figg, Knotts' five-film contract with Universal finished. He continued to work steadily, though he did not appear as a regular on any successful television series until his appearance on Three's Company in 1979. In the late 1960s and early 1970s, Knotts served as the spokesman for Dodge trucks and was featured prominently in a series of print ads and dealer brochures. On television, he went on to host a variety show/sitcom hybrid on NBC, The Don Knotts Show, which aired Tuesdays during the fall of 1970, but the series was low-rated and short-lived. He also made frequent guest appearances on other shows such as The Bill Cosby Show and Here's Lucy. In 1970, appeared as a Barney Fife-like police officer in the pilot of The New Andy Griffith Show. In 1972, Knotts voiced an animated version of himself in two episodes of The New Scooby Doo Movies: "The Spooky Fog of Juneberry", in which he played a lawman resembling Barney Fife, and "Guess Who's Knott Coming to Dinner". He also appeared as Felix Unger in a stage version of Neil Simon's The Odd Couple, with Art Carney as Oscar Madison. Beginning in 1975, Knotts was teamed with Tim Conway in a series of slapstick films aimed at children, including the 1975 Disney film The Apple Dumpling Gang and its 1979 sequel, The Apple Dumpling Gang Rides Again. They also did two independent films, the boxing comedy The Prize Fighter (1979), and the mystery comedy The Private Eyes (1980). Knotts co-starred in several other Disney films, including Gus (1976), No Deposit, No Return (1976), Herbie Goes to Monte Carlo (1977) and Hot Lead and Cold Feet (1978).

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