Cytochrome P450 (P450) peroxygenase reactions are catalyzed by the ferric form of the enzyme and utilize hydroperoxides as oxidant donors. These reactions involve heterolytic cleavage of the hydroperoxide O-O bond and oxidation of the heme iron of P450 to a ferryl-oxyl complex, with subsequent transfer of the activated oxygen to a substrate. In the present study, we have demonstrated that arachidonic acid (AA) can serve as a substrate for P450 peroxygenase activity in the presence of cumene hydroperoxide (CuOOH) as a cosubstrate. AA is transformed into the same primary metabolite classes in both NADPH- and CuOOH-dependent oxidations. However, differences in the efficiency of the formation of the primary metabolite classes by CuOOH suggest that the disposition of AA in the presence of hydroperoxides is highly P450 isozyme dependent. Of particular note was the remarkable efficiency of AA epoxidation observed in the CuOOH-dependent reaction in guinea pig liver, whereas the formation of hydroxylated derivatives of AA at the thermodynamically less reactive positions C16 through C20 was more efficiently catalyzed via NADPH-dependent oxidation. Peroxygenase metabolism of AA and/or lipid hydroperoxide transformation reactions may be important functions for endothelial cells that contain P450 but are deficient in NADPH-P450 reductase, and they may also be involved in the cellular response to oxidative stress during NADPH depletion.