A correlation between the presence of 5.6-kilobase plasmids and bacteriocin activity was found in human-derived strains of Streptococcus mutans. Compared with bacteriocin activity of randomly selected clinical isolates of plasmid-negative strains, bacteriocin activity of plasmid-positive strains significantly inhibited not only two laboratory strains of S. mutans, OMZ176 and AHT (p<0.0001 and p=0.038, respectively), but most plasmid-negative clinical isolates as well (p=0.0005). Comparisons of inhibition between pairs of plasmid-positive strains revealed two groups, group I and group II, that produced distinct bacteriocins we designated as mutacin I and mutacin II, respectively. Within each group, a strain produced inhibitory activity against all the members of the other group but against no members of its own group. Plasmid DNA from plasmid-positive strains of each group was isolated and analyzed by restriction endonuclease digestion. Plasmids from the two groups that were apparently identical in size differed in digestion patterns for EcoRI, HaeIII, and TaqI, even though six TaqI fragments appeared to be common to all. Based on bacteriocin profiles and restriction enzyme digest, two distinct groups of plasmid-positive S. mutans strains emerged. Although the bacteriocin activity of plasmid-positive strain LM7 (serotype e) placed it in group I, clear differences in restriction digests distinguished it from the other plasmid-containing strains.