Selenium-containing glutathione peroxidase (EC 1.11.1.9) was purified 6000-fold from bovine red blood cells to apparent homogeneity. Lipoxygenase (EC 1.13.11.12) was enriched 20-fold from soybean acetone powder. Linoleic acid was peroxidized with lipoxygenase and then used as a substrate in the glutathione peroxidase reaction. Analogous experiments were conducted with synthetic 1,2-dilinoleoyl-L-alpha-glycerophosphocholine and with natural bovine heart cardiolipin. The peroxidized phospholipids were reactive with glutathione peroxidase only after enzymatic attack by phospholipase A2 (EC 3.1.1.4). This result implies that the membrane-protective function of glutathione peroxidase includes preceeding phospholipase action and excludes a direct interaction of this enzyme with membrane-bound lipid hydroperoxides.