Mammalian 15-lipoxygenases have been suggested to be involved in cell differentiation and atherogenesis because of their capability of oxygenating polyenoic fatty acids esterified to biomembranes and lipoproteins. We investigated the interaction of the lipid-peroxidizing 15-lipoxygenase and the hydroperoxy lipid-reducing phospholipid hydroperoxide glutathione peroxidase during their reaction with biomembranes and lipoproteins and obtained the following results. 1) Lipoxygenase treatment of submitochondrial membranes led to the formation of hydroperoxyphosphatidylethanolamine and hydroperoxyphosphatidylcholine as indicated by high performance liquid chromatography with chemiluminescence detection. In 15-lipoxygenase-treated low density lipoprotein cholesteryl hydroperoxylinoleate was the major oxygenation product. 2) Phospholipid hydroperoxide glutathione peroxidase was capable of reducing the hydroperoxy lipids formed by the 15-lipoxygenase to their corresponding alcohols. 3) Preincubation of low density lipoprotein and submitochondrial membranes with the phospholipid hydroperoxide glutathione peroxidase completely prevented the lipoxygenase reaction. However, addition of exogenous hydroperoxy lipids restored the oxygenase activity. 4) Short-term incubations of the complex substrates with the 15-lipoxygenase led to a specific pattern of oxidation products which was rendered more unspecific at long-term incubation or at high substrate concentrations. If the phosholipid hydroperoxide glutathione peroxidase was present during the reaction, the specific product pattern was preserved. These data indicate that the phospholipid hydroperoxide glutathione peroxidase is capable of reducing hydroperoxy ester lipids formed by a 15-lipoxygenase, and that it may down-regulate the 15-lipoxygenase pathways in mammalian cells. The specificity of 15-lipoxygenase-derived hydroperoxy lipids depends on their immediate reduction to the corresponding alcohols preventing postcatalytic isomerization.