Primary cell-mediated microbial mutagenesis assays have been shown to be useful in detecting specific target organ genotoxic activity. The lack of a standard protocol for these assays, however, makes interlaboratory comparisons difficult. In order to standardize the hepatocyte-mediated Salmonella typhimurium mutagenesis assay, incubation and activation conditions for the plate incorporation and preincubation assays were examined using two aromatic amines, 2-aminofluorene (AF) and 2-acetylaminofluorene (AAF). Direct comparison of two preincubation protocols demonstrated the necessity for the hepatocytes to be present during the two-to three-day plate incubation period.