The potent carcinogen benzo[a]pyrene (B[a]P) and its metabolite B[a]P trans-7,8-dihydrodiol (7,8-diol) require metabolic activation by the microsomal cytochrome P450s (P450s) to exert several adverse biological effects, including binding to DNA, toxicity, mutagenicity, and carcinogenicity. In the study reported here, we defined the role of each of 12 individual cDNA-expressed cytochrome P450s in the metabolism of B[a]P and 7,8-diol. Human P450s 1A1 and 1A2 were expressed in the absence or presence of epoxide hydrolase (EH) in a human lymphoblastoid cell line, and six human and five rodent and rabbit P450s were expressed from cDNA with vaccinia virus vectors in the hepatoma cell line Hep G2. B[a]P metabolism resulted in nine metabolites (three diols, three quinones, and three phenols), which were separated, identified, and quantitated by high-pressure liquid chromatography. In the human lymphoblastoid cells, human 1A1 metabolized B[a]P at a rate 4.5 times greater than that for 1A2. EH was shown to be directly involved in B[a]P activation, since increasing the amount of EH resulted in less 7-hydroxybenzo[a]pyrene and more 7,8-diol formation. Of the human P450s expressed with the vaccinia virus vectors in Hep G2 cells, 1A2 and 2C9 showed the highest activity and 2B6 showed moderate activity for B[a]P metabolism. Mouse 1A1 had activity 40 times higher than any human, rabbit, or rodent P450s, indicating the potential pitfalls of extrapolating P450 activity across species. Metabolism of the 7,8-diol resulted in six metabolites (four tetrols and two triols). In the lymphoblastoid cells, human 1A1 was shown to be 4.2 times more active than 1A2 for 7,8-diol metabolism. Among human P450s expressed from vaccinia virus, 1A2, 2E1, and 2C9 gave the highest activity, and 2C8 and 3A4 showed moderate activity for 7,8-diol metabolism to the diol epoxides. Again, mouse 1A1 was much more active than any other P450. These studies, in which we determined the capacity of individual P450 in the metabolism and activation of B[a]P and 7,8-diol, may thus lead to a better understanding of how P450s control the detoxification and activation of polycyclic aromatic hydrocarbons.