A model of lipid peroxidation was employed to investigate the formation of oxidized phospholipids in red blood cell membranes after treatment with t-butylhydroperoxide (tBuOOH). On-line normal-phase HPLC/mass spectrometry (LC/MS) with electrospray ionization was used to separate phospholipid classes and analyze the distribution of the major poly-unsaturated fatty acyl groups and corresponding oxidation products. Arachidonic acid was observed primarily in plasmalogen glycerophosphoethanolamine (GPE), whereas linoleic acid was equally distributed in 1,2-diacyl-GPE and glycero-phosphocholine (GPC) lipids. The additions of one and two oxygen atoms to poly-unsaturated phospholipid molecular species were observed as the major, stable products after incubation with tBuOOH. Tandem mass spectrometry was utilized to further structurally characterize the oxidized fatty acyl groups which were identified as 5-, 8-, 9-, 11-, 12-, and 15-hydroxy-eicosatetraenoate (HETE) and 5-, 12-, and 15-hydroperoxyeicosatetraenoate (HpETE) in addition to 9- and 13-hydroxyoctadecadienoate (HODE) and 9- and 13-hydroperoxyoctadecadienoate (HpODE). Although 18:0p/20:4-GPE was the predominate phospholipid species containing arachidonic acid, the major species containing HETE and HpETE were the 1,2-diacyl-GPE with hexadecanoate as the sn-1 substituent. This result would be consistent with a differential pathway of oxidative degradation of arachidonoyl plasmalogen GPE suggesting a unique role for this plasmalogen molecular species glycerophospholipid.