Abstract Weanling female BALB/c mice were continuously fed diets containing combinations of 12 or 24% protein, 4 or 24% corn oil and 0 or 500 ppm 2-acetylaminofluorene (2-AAF) for approx. 77 weeks to determine the effects of protein, fat and 2-AAF on the biological activity of hepatic microsomal preparations. The S-9 fractions prepared from the livers of the animals were tested in the Ames mutagenicity assay system using Salmonella typhimurium TA-1538 as the indicator strain. The data showed that animals maintained on high fat diets had significantly lower hepatic microsomal activity levels as measured in the in vitro test. In this experiment, the concentration of dietary protein did not affect the activity levels, but 2-AAF was shown to be a strong inducer of hepatic microsomal activity regardless of the other dietary constituents.