Fifty strains of Streptococcus mutans independently isolated from human dental plaque were examined for the presence of covalently closed circular plasmid deoxyribonucleic acid (DNA). Cesium chloride-ethidium bromide centrifugation of [3H]thymidine-labeled, Sarkosyl-lysed cells revealed that 2 of the 50 strains contained plasmid DNA. The plasmid DNA from these strains was characterized by velocity and equilibrium centrifugation and by electron microscopy. The plasmids in these strains were virtually identical in size, with molecular weights of 3.6 X 10(6) and 3.7 X 10(6), Both were present to the extent of approximately 20 molecules per genome equivalent. Interlocked catenated dimeric molecules of each plasmid were readily detected by velocity sedimentation and electron microscopy. These plasmid-containing strains were compared with representative plasmid-free S. mutans strains by using such criteria as bacteriocin production, antibiotic susceptibility, and hemolysis of mammalian erythrocytes. Although no correlation of phenotype to plasmid content could be made, production of bacteriocin-like activity differed significantly between the two plasmid-containing S. mutans isolates. Thus, although the plasmids in these two isolates appeared identical by the criteria of molecular weight, presence of dimers, and copy number, they appeared to be harbored by two distinct S. mutans strains.