AbstractA method is described in which primary rat hepatocytes have been cocultured with Chinese hamster ovary (CHO) cells to provide metabolic activation of promutagens in the Chinese hamster ovary/hypoxanthine‐guanine phosphoribosyl transferase (CHO/HGPRT) mutational assay. Single cell hepatocyte suspensions were prepared from male Fischer‐344 rats using the in situ collagenase perfusion technique. Hepatocytes were allowed to attach for 1.5 hours in tissue culture dishes containing an approximately equal number of CHO cells in log growth. The cocultures were exposed to promutagens for up to 20 hours in serum‐free medium. The survival and 6‐thioguanine‐resistant fraction of treated CHO cells were then determined as in the standard CHO/HGPRT assay. Aflatoxin B1(AFB1) 7,12‐dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene (B(a)P) were found to produce increases in the mutant fractions of treated CHO cells as a function of concentration. The time required for optimum expression of the mutant phenotype following exposure to DMBA and AFB1was approximately 8 days. Primary cell‐mediated mutagenesis may be useful in elucidating metabolic pathways important in the production and detoxification of genotoxic pro