An improved high-pressure liquid chromatography system was used to analyze the amount of benzo[a]pyrene metabolites formed in reconstituted microsomal mixed-function oxidase systems containing different cytochromes P-450. We separated twelve identified and seven unknown metabolites of BP which included three diols: the 9,10-, 4,5-and 7,8-dihydrodiols; four phenols, 9-,7-, 1-, and 3-hydroxybenzo[a]pyrene (OH-BP); and three quinones: the 1,6-. 3,6-, and 6,12-quinones. Two additional peaks co-migrated with synthetic 4-OH-BP and 5-OH-BP, respectively. The former, designated fraction 1, was shown by u.v. spectra to contain primarily the 4,5-epoxide with small amounts of 4-OH-BP. The total metabolism of BP was found to be approximately 20-fold greater with the cytochrome P-450 from the 3-methylcholanthrene (P-450 3-MC) and beta-naphthoflavone (P-450 BNF) treated rats than with the phenobarbital preinduced cytochrome P-450 (P-450 BP). 3-OH-BP ad 9-OH-BP were the major phenolic products for both P-450 3-MC and P-450 BNF whereas the 3-OH-BP and 1-OH-BP were the major phenolic products for P-450 BP. The ratio of total phenols to diols was found to be 3.34, 4.85 and 0.70 for P-450 3-MC, P-450 BNF and P-450 PB. The major dihydrodiol generated by P-450 3-MC and P-450 BNF was 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, whereas the 9,10-diol was the major diol from P-450 PB. The amount of 1,6- and 3,6-quinones produced was greater than the 6,12-quinone with the P-450 3-MC and P-450 BNF but all three quinones were produced in low and equal amounts by the P-450 PB. In respect to the percent metabolites formed at a given region of the BP, P-450 3-MC and P-450 BNF preferred oxidation at the 1, 3 positions, 6 position and the 7, 8 positions, whereas the P-450 PB preferred oxidation at the 4, 5 position. This study demonstrates the unique positional specificity of different forms of cytochrome P-450 which may regulate the balance between activation and detoxification pathways of polycyclic aromatic hydrocarbon metabolism.