This in vitro mutagenicity test system comprises five different strains of S. typhimurium as target cells with the rat liver S-9 fraction and appropriate co-factors for metabolic activation of the chemical tested. The bacterial tester strains detect both mutations induced by base pair substitutions and intercalation (frame shift mutations). Usually 10(8)--10(9) cells of an overnight culture or an exponentially growing culture are incubated for 2-3 days with a mixture of S-9, co-factors, soft agar and the chemical on histidine-deficient agar. The S-9 fraction is obtained from the livers of rats pretreated with 500 mg/kg chlorinated biphenyls (Clophen A-50, Aroclor 1254) to obtain high metabolic activity. For reproducibility it is essential to standardize metabolic activity and protein content of the S-9 and to use three different concentrations thereof in the test system. Since solvents inhibit metabolic activation of the chemicals they must not exceed 4% of the final 2.6 ml incubate. Several independent studies have shown that between 85 and 93% of chemical carcinogens are mutagens in the test. Regarding extrapolation to man one has to consider that the test is preferentially adapted for metabolic activation of the chemicals, whereas inactivation processes are absent or are less active than in vivo. Thus, the test provides qualitative rather than quantitative information on mutagenic effects of a chemical.