When arachidonate 12‐lipoxygenase purified from porcine leukocytes was incubated aerobically with 1‐palmitoyl‐2‐arachidonoyl‐sn‐glycero‐3‐phosphocholine, the phospholipid reacted at up to 30% of the rate of a free fatty acid substrate; the esterified arachidonic acid was oxygenated predominantly to the (12S)‐12‐hydroperoxy product. The porcine leukocyte enzyme was also capable of metabolizing phosphatidylcholine containing esterified (15S)‐15‐hydroperoxy‐5,8,11,13‐eicosatetraenoic acid; oxygenation occurred predominantly at the 14R position. Reaction with mitochondrial and endoplasmic membranes of rat liver produced esterified (12S)‐12‐hydroperoxy‐5,8,10,14‐eicosatetraenoic acid and (13S)‐13‐hydroperoxy‐9,11‐octadecadienoic acid as major oxygenation products. Thus, porcine leukocyte 12‐lipoxygenase is capable of oxygenating not only free polyenoic fatty acids but also more complex substrates such as phospholipids and biomembranes. In contrast, the human platelet 12‐lipoxygenase is almost inactive with these esterified polyenoic fatty acids. In regard to the function of these enzymes, the leukocyte‐type of 12‐lipoxygenase has similar catalytic activities to the mammalian 15‐lipoxygenase and its physiological function may include the structural modification of membrane lipids.