In the present study, phospholipase A(2) (PLA(2))-catalyzed hydrolysis of platelet membrane phospholipids was investigated by measuring PLA(2) activity, phospholipid hydrolysis, arachidonic acid release and choline lysophospholipid production in thrombin-stimulated human platelets. Thrombin-stimulated platelets demonstrated selective hydrolysis of arachidonylated plasmenylcholine and plasmenylethanolamine, with little change in diacyl phospholipids. Accelerated plasmalogen hydrolysis was accompanied by increased arachidonic acid and thromboxane B(2) release and increased lysoplasmenylcholine production. Thrombin stimulation caused an increase in PLA(2) activity measured in the cytosolic fraction with plasmenylcholine only; no increase in activity was measured with phosphatidylcholine. No change in membrane-associated PLA(2) activity was observed with either substrate tested. Pretreatment with the Ca(2+)-independent PLA(2)-selective inhibitor, bromoenol lactone, inhibited completely any thrombin-stimulated phospholipid hydrolysis. Thus, thrombin stimulation of human platelets activates a cytosolic PLA(2) that selectively hydrolyzes arachidonylated plasmalogen phospholipids.