By chromatographic and mass spectral techniques we document in rat liver the presence of new classes of glycerophospholipids which contain an epoxyeicosatrienoate moiety, esterified to the glycerol-sn-2 position. These novel lipids are formed in vivo from endogenous precursors and under physiological conditions. Chromatographic resolution followed by hydrolysis and regioisomeric analysis showed that they consist of the 8,9-, 11,12-, and 14,15-epoxyeicosatrienoyl derivatives of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. Their relative concentrations (micromoles of oxidized lipid/mol of phospholipid) were 70, 85, and 106 for phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol, respectively. Chiral analysis of the fatty acids at sn-2 revealed an enantioselective preference for 8(S),9(R)-, 11(S),12(R)-, and 14(R),15(S)-epoxyeicosatrienoates in all three lipid classes. The data suggest a multienzyme process initiated by cytochrome P-450 epoxidation of arachidonic acid followed by ATP-dependent activation to epoxyeicosatrienoyl-CoA derivatives and stereoselective lysolipid acylation. These results provide a molecular basis for a potential physiological role of cytochrome P-450 in the biosynthesis of unique cellular glycerolipids and, consequently, in the control of cell membrane structure and function.