After garrison duty at the United States Naval Academy in Annapolis, Maryland, the regiment served with the Coast Division commanded by Maj. Gen. Ambrose Burnside. The Coast Division was deployed in January 1862 for operations on the coast of North Carolina, and participated in the Battle of Roanoke Island and the Battle of New Bern among other engagements. Burnside's division was recalled to Virginia in July 1862. The 21st Massachusetts Infantry was then attached to the Army of the Potomac and participated in several of the largest battles of the Civil War, including the Second Battle of Bull Run, the Battle of Antietam, and the Battle of Fredericksburg. The most devastating engagement of the war for the 21st was the Battle of Chantilly, fought on September 1, 1862, during which the unit suffered 35 percent casualties. From March 1863 to January 1864, the 21st served with Burnside in the Department of the Ohio, seeing action in Kentucky and eastern Tennessee. In May 1864, the regiment rejoined the Army of the Potomac, participating in Lt. Gen. Ulysses Grant's Overland campaign and the siege of Petersburg. The regiment was a favorite of Clara Barton, the famed battlefield nurse, who was also from Worcester County, Massachusetts.