Primary cultures of adult rat parenchymal hepaiocytes were developed as an in vitro model to investigate the biochemical fate of 2‐acetylaminofluorene (AAF), a potent hepatocarcinogen. More than 5 × 108viable cells were routinely isolated by collagenase perfusion of 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 μg AAF was metabolized in 21–24 h by 2 × 106cultured 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 of 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 10−4M SKF 525A and 100% with 10−3M SKF 525A. At a dose of 40 μg AAF per 2 × 106cells, only 31% of the carcinogen was recovered from the culture medium as water‐soluble products after 24 h; the cells were shown to be capable of metabolizing a subsequent 40‐μg dose at an un‐diminished 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.