The various properties of the inhibitory substances produced by Streptococcus mutans strains C67-1, Ny257-S, Ny266, and T8, and the fact that inhibitory zones produced could not be associated with lactic acid (or other organic acids), bacteriophages, or hydrogen peroxide indicate that these substances can be classified as mutacins. The substances produced by strains C67-1, Ny266, and T8 possessed similar properties. They were shown to be thermoresistant (100 degrees C, 30 min), low molecular weight (less than 3500) substances sensitive to proteolytic enzymes (chymotrypsin, papain, pronase E, proteinase K, and trypsin) and they were active against most of the Gram-positive bacteria tested but not against most of the Gram-negative bacteria. The substance produced by strain Ny257-S differs from the other three by its narrower activity spectrum, its lower thermoresistance (80 degrees C, 30 min), and its higher molecular weight (8,000-14,000). The gene or the genes coding for the mutacins are probably located on the chromosome since no plasmid DNA could be detected in these four producing strains. Restriction-fragment patterns of C67-1 and T8 suggest that these strains are closely related, as supported by the strong similarity observed between the properties of their mutacins.