The effect of bacteriocins on the composition of dental plaque flora was studied in vitro with bacterial plaque formed by oral streptococci on glass rods suspended in broth medium. Cell-free preparations containing mutacin SW31, a bacteriocin produced by Streptococcus mutans , killed sensitive cells present in the plaque selectively, but did not affect resistant cells. Similar preparations from a non-bacteriocinogenic mutant exerted only a slight effect. Mixed growth of bacteriocin-producing S. mutans SW31 with the sensitive S. sanguis Ny101 resulted in a nearly single-strain plaque of the former. Mutacin could be detected in the plaque substance as well as in the surrounding medium. A non-bacteriocinogenic mutant, which was shown not to be altered in functions affecting growth in plaque, allowed substantial growth of S. sanguis Ny101. Sequential inoculation of the bacteriocinogenic plaque former S. sanguis P3A3 and the sensitive S. mutans OMZ61 showed that the cells of the latter are killed rapidly in established plaques when inoculated with the bacteriocinogenic strain. A trypsin-sensitive antagonistic substance could be detected in the plaque, but not in the surrounding medium. The results indicate that bacteriocins can be active in plaque in vitro and suggest that bacteriocins could play a role in determining the composition of plaque in vivo.