Exogenous unlabeled arachidonic acid (AA) added to human platelets prelabeled with [3H]AA induces breakdown of [3H]phosphatidylinositol and the rapid and transient formation of [3H]1,2-diacylglycerol and [3H]phosphatidic acid (PA), indicating activation of phosphatidylinositol-specific phospholipase C. Formation of [3H]1,2-diacylglycerol and [3H]PA is inhibited by pretreatment of platelets with aspirin, which suggests that endoperoxides or thromboxane A2 are responsible for AA-induced stimulation of phospholipase C. Exogenous unlabeled AA also induces the formation of [32P]PA or [14C]PA in platelets that have been prelabeled with 32Pi or [14C]AA, respectively. Increased radioactivity in PA reflects increased content of PA as measured by the fatty acid composition of PA. The relation of PA production, which reflects stimulation of phospholipase C, to specific platelet responses was further investigated. Low concentrations of AA (0.05-0.2 microM) induces platelet shape change in parallel to formation of 50-100% PA and phosphorylation of a 40,000 molecular weight protein. Higher concentrations of AA (0.5-50 microM) stimulate the formation of a further amount of PA (200-250%), and phosphorylation of 40,000 molecular weight protein, platelet aggregation, and serotonin release. Indomethacin inhibits all these observed changes by inhibiting the conversion of AA by platelet cyclooxygenase. In contrast, prostacyclin blocks these responses without affecting conversion of AA by platelet cyclooxygenase and thromboxane synthetase. We conclude that formation of endoperoxides and thromboxane A2 is necessary but not sufficient for platelet activation by AA. Only if PA is formed are platelets activated. The results indicate a central role for the phospholipase C pathway in the process of platelet activation.