Abstract Reconstituted nonfat dry milk was fermented by Lactobacillus helveticus CH65, Lactobacillus acidophilus BG2FO4, Streptococcus salivarius ssp. thermophilus CH3, Lactobacillus delbrueckii ssp. bulgaricus 191R, and by a mixture of the latter two organisms. The fermented milks were then freeze-dried, extracted in acetone, dissolved in dimethylsulfoxide, and assayed for antimutagenicity in the Ames test (Salmonella typhimurium TA 100) against N-methyl, N’-nitro, N-nitroso-guanidine, and 3,2’-dimethyl-4-amino-biphenyl. Dose-dependent activity was significant against both mutagens in all extracts. Maximal inhibitory activity against 3,2’-dimethyl-4-aminobiphenyl and N-methyl, N’-nitro, N-nitroso-guanidine was 2- and 2.7-fold greater, respectively, than that exhibited by extracts of unfermented milk. Extracts of milk fermented by L. delbrueckii ssp. bulgaricus 191R were examined further. Compounds that were responsible for activity against both mutagens were less soluble in aqueous solutions than in dimethylsulfoxide. Adjustment of milk fermented by L. delbrueckii ssp. bulgaricus 191R to pH 3, 7.6, or 13 prior to freeze-drying and acetone extraction did not significantly alter the activity specific for 3,2’-dimethyl-4-aminobiphenyl. In contrast, compounds with activity specific for N-methyl, N’-nitro, N-nitrosoguanidine were less extractable at pH 7.6. The weak antimutagenicity of unfermented milk was not increased by addition of 2% L-lactic acid.