Metabolites of benzo(e)pyrene (B[e]P) formed upon incubation of [3H]-B[e]P with hepatic microsomes from control and induced rats have been separated by high-pressure liquid chromatography and identified by comparison of retention times, absorbance and fluorescence spectra with those of synthetic standards. The major metabolite produced was B[e]P-4,5-dihydrodiol, accounting for 20-30% of the total metabolism depending on the source of the microsomes. This was followed by a phenolic metabolite (shown not to be 4-OH-; 9-OH-; or 10-OH-B[e]P). A possible proximate carcinogenic derivative of B[e]P, B[e]P-9,10-dihydrodiol, was identified, but was found to constitute less than 1% of the total metabolites. Similar results were obtained with a purified and reconstituted mixed-function oxidase system. In these later incubations, production of the dihydrodiols was dependent on the addition of purified epoxide hydrase to the incubation mixtures. These results suggest that formation of the reactive diol-epoxide, 9,10-dihydroxy-11,12-epoxy-9,10,11,-12-tetrahydro-B[e]P, a potential ultimate carcinogenic metabolite of B[e]P, is not favored by rat liver enzymes. This provides a partial explanation for the lack of carcinogenicity of B[e]P within the framework of the bay region theory of chemical carcinogenesis.