The products formed in the metabolism of 3-methylcholanthrylene (3MCE), either in the presence or in the absence of an epoxide hydrolase inhibitor, 3,3,3-trichloropropylene 1,2-oxide (TCPO), with an NADPH-regenerating system and liver microsomes from 3-methylcholanthrene (3MC)-treated male Sprague-Dawley rats were separated by reversed-phase and normal-phase HPLC. The metabolites were characterized by UV-visible absorption spectral analysis, and by comparing their retention times on reversed-phase and normal-phase HPLC with authentic 3MC derivatives whenever available. In addition to 3MC trans-1,2-diol, 3MC-1-one, and 3MC-2-one reported earlier by other investigators, 3-hydroxymethylcholanthrylene (3-OHMCE), 3-OHMCE trans-11,12-dihydrodiol, 3MCE trans-11,12-dihydrodiol, 3MCE trans-9, 10-dihydrodiol. 9- and 10-hydroxy-3MCE. 3MC-2-one trans-9,10-dihydrodiol, and a chemically unstable 3MCE 1,2-epoxide were identified as metabolites of 3MCE. 3MC cis-1,2-diol, a previously reported metabolite of 3MCE, was not detectable. In the presence of TCPO, metabolites that have been identified include 3-OHMCE, 3-OHMCE 11,12-epoxide. 3MCE 11,12-epoxide, 3MC-2-one, 3MC-1-one, 9-hydroxy-3MCE, 10-hydroxy-3MCE, and an unstable metabolic intermediate 3MCE 1,2-epoxide. The results suggest that 3MCE 1,2-epoxide, 3MCE 9,10-diol-7,8-epoxide, and 3MC-2-one 9,10-diol-7,8-epoxide may be involved in the metabolic activation of 3MCE to carcinogenic form.