beta-Lactamase encoded by a small, nontransferring R-plasmid, NTP1, conferring ampicillin resistance to its host bacteria, was purified. NTP1 plasmid-coded beta-lactamase was found to be periplasmically located in the host Escherichia coli cell, to have a molecular weight of about 25,000, and to show a relatively low activity against oxacillin and methicillin compared with benzylpenicillin. These characteristics indicate that NTP1 plasmid-coded beta-lactamase is very similar or identical to the "TEM-type" beta-lactamase, which is the most common beta-lactamase coded by R-plasmids in enteric bacteria. In minicells containing NTP1 plasmids, at least six plasmid-specific proteins were synthesized, and beta-lactamase was synthesized in a greater amount than other plasmid-coded proteins. In a cell-free transcription-translation coupled system from E. coli, NTP1 plasmid deoxyribonucleic acid directed the synthesis of several species of plasmid-specific proteins, including active beta-lactamase. The in vitro system also showed preferential synthesis of beta-lactamase, as was observed in minicells containing NTP1 plasmids.