A novel ACTH-inducible P450, cytochrome P450RAP, is responsible for polycyclic aromatic hydrocarbon (PAH) metabolism in male rat adrenal microsomes. P450RAP is present at similar levels in male and female adrenal microsomes and is immunochemically distinct from P450IA1. Anti-P450RAP immunoblots a protein present in ovarian and testicular microsomes that is the same size as P450RAP and which coelutes with the P450 fraction during chromatography on an immobilized artificial membrane column made with phosphatidylcholine. Rat adrenal, ovarian, and testicular microsomes exhibit similar regioselectivities in the metabolism of two polycyclic aromatic hydrocarbons, dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene (BP). Unlike P450IA1, these microsomes form little or no 7-OH-DMBA and BP-4,5-diol but do catalyze the formation of a high proportion of the presumptive procarcinogen, DMBA-3,4-diol. The relative activities of PAH metabolism by untreated adrenal, testicular, and ovarian microsomes are approximately 60, 20, and 6 pmol/mg microsomal protein/min, respectively. PMSG induced PAH metabolism 2- to 5-fold in ovarian microsomes and also increased the P450RAP immunoblot. Hypophysectomy reduced PAH metabolism 3-fold in testicular microsomes while also decreasing the P450RAP immunoblot. This close correlation between PAH metabolism and expression of P450RAP indicates the involvement of the cytochrome in this activity. DMBA and BP metabolism by PMSG-treated rat ovarian microsomes and untreated testicular microsomes are each completely inhibited by anti-P450RAP but are not inhibited by anti-P450IA1. Essentially all of the PAH metabolism in rat adrenal, testis, and ovary is, therefore, catalyzed by P450RAP, which is hormonally elevated in each tissue by a variety of possible mechanisms, including induction and selective proliferation of cells that express this protein.