The release of arachidonic acid, and its subsequent conversion to thromboxane A2, is an important component of platelet activation. The precise mechanism of arachidonic acid release is unknown although cytosolic phospholipase A2 (cPLA2) has been implicated. In the present study the effects of three agonists, the serine protease thrombin, the protein kinase C stimulant PMA and the calcium ionophore A23187 have been examined on the translocation and phosphorylation of cPLA2 and these have been correlated with arachidonic acid release. Thrombin, but neither PMA nor A23187, caused the release of [14C]-arachidonic acid from unstirred, prelabeled platelets. Immunoblot analysis was carried out on cytosolic and membrane fractions from control and activated platelets using an anti-cPLA2 antibody. In platelets stimulated by thrombin or A23187, but not by PMA, there was a translocation of cPLA2 to the membrane fraction. Immunoprecipitation of cPLA2 from [32P]-ortho-phosphate-prelabeled platelets, indicated enhanced phosphorylation on serine residues of cPLA2 from thrombin- or PMA-stimulated platelets. These results are consistent with two synergistic pathways mediating cPLA2 activity. Increased cytosolic calcium causes the translocation of cPLA2 to the membrane, and protein kinase either directly, or indirectly, phosphorylates the enzyme. Activation of both pathways, as occurs in response to thrombin, is required for arachidonic acid liberation.