The major oxidation product of the classic polycyclic hydrocarbon carcinogen benzo(a)pyrene [B(a)P] is 3-hydroxy B(a)P. Numerous studies have been concerned with the measurement of B(a)P 3-hydroxylation activity in experimental animals and human tissues. Although human liver is the main site of this reaction, systematic studies had not been carried out to define the roles of individual cytochrome P-450 (P-450) enzymes involved. Purified human P4502C8 and P4503A4 showed appreciable catalytic activity; purified human P4501A2 and yeast recombinant (human) P4502C9 and P4502C10 had less activity. No B(a)P 3-hydroxylation activity was observed with purified human P4502A6, P4502D6, P45602E1, or P4502CMP. When microsomes prepared from different human liver samples were compared, B(a)P 3-hydroxylation activity was well correlated with nifedipine oxidation (a P4503A4 marker) but not markers of other P-450s, including tolbutamide hydroxylation (P4502C9 and 2C10), chlorzoxazone 6-hydroxylation (P4502E1), (S)-mephenytoin 4'-hydroxylation (P4502CMP), and coumarin 7-hydroxylation (P4502A6). In three of the liver microsomal samples with relatively high B(a)P 3-hydroxylation activity, immunoinhibition was observed with anti-P4503A greater than anti-P4502C (and no inhibition with several other antibodies). The selective chemical inhibitors gestodene and troleandomycin (P4503A enzymes) and sulfaphenazole (P4502C enzymes) reduced the B(a)P 3-hydroxylation activity of the more active microsomal preparations to rates seen in the preparations with low activity. This residual activity (and most of the activity in the low activity samples) was refractory to all of the chemical inhibitors and antibodies. The addition of 7,8-benzoflavone dramatically stimulated B(a)P 3-hydroxylation in all of the microsomal samples (and also stimulated purified P4503A4), arguing against an important role for P4501A1 or P4501A2. We conclude that roles of human P-450 enzymes for B(a)P 3-hydroxylation follow the order P4503A4 greater than or equal to P4502C8 greater than P4502C9/10 in human liver and that the other P-450s examined here do not have major roles. P4502C8 and P4502CMP (but not P4503A4) were found to activate B(a)P to products genotoxic in Salmonella typhimurium; this pathway would appear to involve products other than 3-hydroxy B(a)P and B(a)P 7,8-dihydrodiols.