Formation of four oxidative metabolites from the anticonvulsant drug phenytoin (DPH) catalyzed by human liver microsomal cytochrome P450 (P450) enzymes was determined simultaneously. Under the conditions in which linearity for formation of 4'-hydroxylated DPH (4'-HPPH; main metabolite) was observed, human liver cytosol increased microsome-mediated DPH oxidation. 3',4'-Dihydroxylated product (3', 4'-diHPPH) formation was 10 to 40% of total DPH oxidation in the presence of liver cytosol. 3'-Hydroxy DPH formation was catalyzed by only one of the human liver microsomal samples examined and 3', 4'-dihydrodiol formation could not be detected in all samples. In the presence of liver cytosol, 3',4'-diHPPH formation activity from 100 microM 4'-HPPH was correlated with testosterone 6beta-hydroxylation activity and CYP3A4 content. However, 3', 4'-diHPPH formation using 1 or 10 microM 4'-HPPH as a substrate was not correlated with contents of any P450s or marker activities. Of 10 cDNA-expressed human P450 enzymes examined, CYP2C19, CYP2C9, and CYP3A4 catalyzed 3',4'-diHPPH formation from the primary hydroxylated metabolites (3'-hydroxy-DPH and 4'-HPPH). Fluvoxamine and anti-CYP2C antibody inhibited 3',4'-diHPPH formation from 10 microM 4'-HPPH in a human liver sample that contained relatively high levels of CYP2C, whereas ketoconazole and anti-CYP3A antibody showed inhibitory effects on the activities in liver microsomal samples in which CYP3A4 levels were relatively high. These results suggest that CYP2C9, CYP2C19, and CYP3A4 all have catalytic activities in 3',4'-diHPPH formation from primary hydroxylated metabolites in human liver and that the hepatic contents of these three P450 forms determine which P450 enzymes play major roles of DPH oxidation in individual humans.