Primary cultures of adult rat parenchymal hepatocytes were developed as an in vitro model to investigate the biochemical fate of 2-acetylaminofluorene (AAF), a potent hepatocarcinogen. More than 5 x 10(8) viable cells were routinely isolated by collagenase perfusion in rat liver; the cells were cultured 2-5 d on collagen-coated dishes in serum-free culture medium containing hormones and other factors to retard the decline of cytochrome P-450. All of 137 ng or 13.7 microgram AAF was metabolized in 21-24 h by 2 x 10(6) cultured hepatocytes in 4.0 ml defined medium. At the higher dose, water-soluble metabolites appeared at 70% of the rate of metabolism at the lower dose, which was 17 ng/h for the initial 4 h. As the parent compound was consumed, bound AAF residues were recovered with exhaustively extracted, trichloro-acetic acid-precipitated hepatocellular macromolecules, accounting for a maximum of 5% of the 137-ng dose. Addition of hormones to the culture medium stimulated the rate of appearance of water-soluble metabolites, AAF, correlating with the enhanced cytochrome P-450 levels of hormone-treated cells. Metabolism of AAF was diminished 50% during 3 h of incubation with 10(-4) M SKF 525A and 100% with 10(-3) M SKF 525A. At a dose of 40 microgram AAF per 2 X 10(6) cells, only 31% of the carcinogen was recovered from the culture medium as water-soluble products after 24 h; the cells were sown to be capable of metabolizing a subsequent 40-microgram dose at an undiminished rate, suggesting that saturation of metabolizing enzymes rather than toxicity occurred. These results support the validity of primary hepatocyte cultures as a model system for quantitative investigations of the biochemical fate of AAF in mammalian cells, and provide preliminary characterization of the cells' processes of detoxification and metabolic activation of a chemical carcinogen.