A microsome-mediated mutagenesis system has been established with the V79 Chinese hamster cell line. The cells, grown in monolayer, were treated with various nitrosamines in the presence of a postmitochondrial fraction ((S15) from rat liver and a reduced nicotinamide adenine dinucleotide-generating system for 1 hr, washed and incubated for 2 to 3 hr in fresh culture medium, and then plated for toxicity and mutagenicity assays. Mutation was determined by resistance to 20mug 8-azaguanine per ml. In this assay system, the S15 fraction and cofactors by themselves were not toxic to the cells; dose-related mutagenicity and cytotoxicity were induced by N-nitrosodimethylamine (DMN) only in the presence of the S15 fraction and cofactors. Pretreatment of rats with phenobarbitone led to an approximately 2-fold increase in the mutation rate over that with tissues from untreated rats with concentrations of DMN from 10 to 50 mM, while aminoacetonitrile pretreatment reduced the mutagenic effect. Methylcholanthrene pretreatment resulted in an increase in the mutation frequency with a higher concentration of DMN (50mM). Various other nitrosamines were also assayed in the presence or absence of the S15 fraction from phenobarbitone-pretreated rats and a reduced nicotinamide adenine dinucleotide phosphate-generating system. With the exception of N-nitrosomethylphenylamine, the carcinogenic nitrosamines (DMN, N-nitrosodiethylamine, N-nitrosodi-n-propylamine, N-nitrosodi-n-butylamine, N-nitrosodi-n-pentylamine, N-nitrosomethyl-n-propylamine, N-nitrosomorpholine, N-nitrosopyrrolidine, N-nitroso-N'-methylpiperazine, and N-nitrosomethylphenylamine) were mutagenic to the V79 Chinese hamster cells in the presence of the S15 fraction and cofactors. Neither N-nitrosodiphenylamine nor N-nitrosomethyl-tert-butylamine had a mutagenic effect. These findings show that chemical carcinogens can be tested for mutagenicity in cultured mammalian cells in the presence of a metabolic activation system. The results are discussed in relation to the carcinogenicity and mutagenicity of these compounds in other test systems.