Benzo[a]pyrene (BaP) metabolism was studied in cell lines derived from rainbow trout (RTG-2), bluegill fry (BF-2), and fathead minnow (FHM). Confluent cultures were exposed to 3H-BaP (0.5 nmol/ml), and, after various exposure times, metabolites were extracted from the media with an organic solvent and analyzed by high-pressure liquid chromatography. BF-2 and RTG-2 cells converted 63% of the BaP to water-soluble metabolites within 24 h, while FHM cells converted only 12%. BF-2 and RTG-2 cells metabolized more than 90% of the BaP by 48 h, while only 67% of the BaP was converted to water-soluble metabolites by FHM cells after 96 h. The major organic-solvent-extractable metabolites in all three cell lines were 9,10-dihydroxy-9,10-dihydrobenzo[a]pyrene and unidentified polar metabolites. Of the water-soluble metabolites formed by BF-2, FHM, and RTG-2 cells, 67, 42, and 19%, respectively, were converted to ethyl-acetate-extractable metabolites by treatment with beta-glucuronidase. All three cell lines formed a glucuronide of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (7,8-diol); in BF-2 and FHM cells, the 7,8-diol represented almost half of the metabolites released by beta-glucuronidase treatment. Thus, cell lines derived from three widely distributed species of freshwater fish have the capacity to metabolize BaP to a form that is a proximate carcinogen in rodents and to produce a water-soluble conjugate of this metabolite.