Substrate specificity of platelet phospholipase A2 was investigated following Ca2+-dependent hydrolysis by endogenous enzyme of linoleate- or arachidonate-labelled platelet phospholipids. Alkylacyl, alkenylacyl and diacyl classes of ethanolamine and choline glycerophospholipids (GPE and GPC) were separated after their diacylglycerol derivation, and molecular species of diacyl-GPE were analyzed by HPLC. Hydrolysis of platelet ethanolamine and choline glycerophospholipids was dependent on Ca2+ and was maximal at neutral pH. In the presence of 0.2 mM Ca2+ the hydrolysis rate for [14C]arachidonate-labelled phospholipids was in the order diacyl-GPE greater than alkylacyl-GPE = diacyl-GPC = alkenylacyl-GPE greater than alkylacyl-GPC. In addition to being the best substrate at high Ca2+ concentration, diacyl-GPE could be degraded with Ca2+ concentrations in the micromolar range, concentrations which are unable to induce any degradation of diacyl-GPC. As a function of Ca2+ concentration, the hydrolysis rate of [14C]linoleate- and [14C]arachidonate-labelled diacyl-GPE or diacyl-GPC was identical. The five main molecular species of diacyl-GPE labelled with arachidonate or with linoleate were hydrolyzed at the same rate in the presence of 50 microM Ca2+. This study shows that platelet phospholipase A2 is specific for endogenous diacyl-GPE and is independent of fatty chain composition. These results are discussed in relation to the Ca2+ concentration observed in stimulated platelets and in relation to the lysophospholipid-induced specific transfer of arachidonate. They suggest that diacyl-GPE hydrolysis by phospholipase A2 could play a key role in stimulated platelets.