The metabolism of platelet-activating factor (PAF) and arachidonic acid is linked through the common intermediate 1-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine (alkylarachidonoyl-GPC). Hydrolysis of alkylarachidonoyl-GPC by phospholipase A2 may initiate the biosynthesis of both PAF and eicosanoids, since alkyllyso-GPC is formed for acetylation to PAF and arachidonic acid is liberated for conversion to biologically active metabolites. In order to elucidate the regulation and functional role of human platelet phospholipase A2 in the pathway leading to the formation of both classes of lipid mediators, we have characterized its action upon alkylarachidonoyl-GPC. Human platelet phospholipase A2 was solubilized and then partially purified in the presence of n-octyl-beta-D-glucopyranoside (octyl glucoside). Hexadecylarachidonoyl-GPC was prepared biosynthetically using platelet sonicates, purified by two-step high-performance liquid chromatography (HPLC) and suspended in buffer by sonication. Our results indicate that deacylation of alkylarachidonoyl-GPC by platelet phospholipase A2 has an absolute requirement for Ca2+. It occurs at submicromolar concentrations of free Ca2+ and exhibits a biphasic Ca2+-dependence with activity plateaus at 10 microM and 2 mM. Phospholipase A2-mediated hydrolysis of alkylarachidonoyl-GPC is increased 2-fold by albumin and is enhanced 5-fold if 1,2-dioleoylglycerol is incorporated into the substrate dispersion. The substrate dependence and specificity of platelet phospholipase A2 for 1-alkyl- vs. 1-acyl-linked subclasses of arachidonic acid containing phosphatidylcholine was examined with 1-O-hexadecyl-2-arachidonoyl-sn-glycero-3-phosphocholine (hexadecylarachidonoyl-GPC) and 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (palmitoylarachidonoyl-GPC). We found that the substrates were deacylated equivalently. We conclude that, in stimulated platelets, in the presence of increased levels of cytoplasmic free Ca2+ and newly generated diacylglycerol, alkylarachidonoyl-GPC may be rapidly hydrolyzed by phospholipase A2 and may serve as a precursor of both PAF and eicosanoids.