The coumarin derivative, cloricromene, an antithrombotic drug previously indicated as AD6, is known to inhibit the release of radioactive arachidonic acid from human platelets prelabelled with arachidonic acid and stimulated with thrombin. This effect might be due to the drug itself or to its catabolite, cloricromene acid. When added to platelet lysates neither compound inhibited phospholipase A2 activity assayed either with endogenous or with exogenous substrates. However, some inhibition was instead shown when intact platelets were first exposed to cloricromene and then enzyme activity was assayed in the lysate. Preincubation of platelets with the drug caused a dose-dependent inhibition of arachidonic acid mobilization in fluoroaluminate-stimulated platelets. beta-Thromboglobulin (beta-TG) release, a phenomenon previously shown to share common steps with phospholipase A2 activation, was also dose-dependently inhibited by cloricromene. Cloricromene also reduced the radioactivity associated with phosphatidic acid in fluoroaluminate-stimulated platelets but not in platelets stimulated with thrombin. These results are consistent with the hypothesis that cloricromene, or its catabolite, inhibits the production of arachidonic acid in stimulated platelets by interfering with a G-protein mediated activation of phospholipase A2 that is independent from the receptor-activated phosphoinositide phospholipase C.