A Tween 20-solubilized preparation of prostaglandin endoperoxide synthase has been shown to metabolize 7,8-dihydrobenzo[a]pyrene (H2BP) to a form highly mutagenic to Salmonella typhimurium strain TA98. The arachidonic acid-dependent metabolism of H2BP by microsomal and purified prostaglandin endoperoxide synthase has been studied and the products identified. A spectral investigation of the metabolism indicated the bay-region double bond as the primary site of metabolism. Radiolabeled H2BP was synthesized and incubated with the enzyme preparations and the metabolites were separated by reverse phase high performance liquid chromatography and quantitated by liquid scintillation counting. Radioactive products were characterized by co-chromatography with chemically synthesized standards, UV-visible spectra, and mass spectrometry of acetate derivatives. The major polar products were determined to be trans- and cis-9,10-dihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene and 7,8,9,10-tetrahydrobenzo[a]pyrene-9-one in a ratio of 1:1.2:0.4. The inclusion of 5 mM 3,3,3-trichloropropene-1,2-oxide, an epoxide hydrolase inhibitor, produced the same products but in a ratio of 1:2.3:1.2. Incubations with purified prostaglandin endoperoxide synthase yielded the three products in a ratio of 1:2.8:0.7. The major nonpolar product was identified as benzo[a]pyrene. The polar products of metabolism, the effects of 3,3,3-trichloropropene-1,2-oxide on their distribution, and the detection of a mutagenic intermediate support the conclusion that H2BP is co-oxygenated during prostaglandin biosynthesis to 9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene.