The role of phospholipase A2 (PLA2) in the formation of platelet-activating factor (PAF-acether) by rabbit platelets is supported by several pieces of evidence. The release of PAF-acether was accompanied by that of lyso-PAF-acether. EDTA, EGTA [ethylene-glycol-bis-(.beta.-aminoethyl ether)N,N''-tetraacetic acid], dibutyryl cAMP, p''-bromophenacylbromide and 874 CB, which, in spite of their structural diversity, are all PLA2 blockers, inhibited the release of both PAF-acether and the lyso-compound. Addition of hog pancreas PLA2 to platelets as well as platelet lysis resulted in the release of lyso-PAF-acether, thus mimicking the metabolic events initiating formation of PAF-acether. PLA2 activation may trigger both the 2nd and the 3rd pathway of platelet activation.