Stimulation of rabbit platelets with U46619 as well as thrombin significantly increased cytosolic phospholipase A2 activity. However, arachidonic acid liberation in response to U46619 was slight compared with that evoked by thrombin. Ionomycin enhanced arachidonic acid liberation induced by thrombin, but had no effect on that by U46619, although ionomycin further increased both thrombin- and U46619-induced Ca2+ mobilization. However, stimulation with U46619 followed by addition of NaF plus AlCl3 synergistically induced arachidonic acid liberation. These results suggest that phosphorylation and translocation to membranes of cytosolic phospholipase A2 are necessary but not sufficient for arachidonic acid liberation by the enzyme upon receptor stimulation, and that another factor, such as GTP-binding protein, may be further required for the hydrolytic action of the enzyme.