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. An examination of various preincubation times showed relatively minor differences between 15 and 90 minutes. The preincubation and plate incorporation protocols were directly compared using both hamster and rat hepatocytes.