Oxygenation of lysophospholipids with unsaturated fatty acyl chains, 1-linoleoyl, 1-arachidonoyl or 1-docosahexaenoyl group by mammalian lipoxygenase (LOX), reticulocyte or leukocyte LPO, was examined by monitoring the formation of conjugated dienes. In support of the above, the hydroperoxy derivative of each lysophospholipid as oxygenation product was confirmed by LC/MS analyses. Further, the oxygenation products of lysophospholipids were found to contain hydroperoxide group mainly at C-13 and C-15 for linoleoyl and arachidonoyl chain, respectively. The kinetic study indicated that lysophosphatidylcholine and lysophosphatidic acid containing polyunsaturated fatty acyl chains were no less efficient than the corresponding polyunsaturated fatty acids as substrate of reticulocyte LOX. Similarly, these lysophospholipids were also found to be oxygenated effectively by porcine leukocyte LOX. In contrast, these lysophospholipids were not oxygenated efficiently by potato LOX. Thus, polyunsaturated lysophospholipids could be utilized as selective substrates for some mammalian lipoxygenases.