Thrombin-induced changes in arachidonate content of platelet phospholipids were quantitated to establish the ultimate origins of this eicosanoid precursor. Fifteen seconds following thrombin addition (15 U/5 X 10(9) platelets), phosphatidylcholine lost 11.8 nmol of arachidonate and phosphatidylethanolamine lost 10.5 nmol. Arachidonate in phosphatidate, phosphatidylinositol, and phosphatidylinositol-4,5-bisphosphate combined decreased by 11.0 nmol. Increases in free and oxygenated arachidonate (41 nmol) exceeded decreases in inositides. Thus phospholipase A2 released at least twice as much arachidonate as phospholipase C-diglyceride lipase. Phosphatidylinositol-4-phosphate levels remained unchanged upon stimulation. Therefore, increases in phosphatidylinositol-4,5-bisphosphate indicated the minimum rate of phosphorylation of phosphatidylinositol to resynthesize phosphatidylinositol-4,5-bisphosphate, following stimulus-induced breakdown by phospholipase C. Phosphatidylinositol-4, 5-bisphosphate increased 1.4 nmol between 10 and 15 sec following thrombin, markedly less than phosphatidylinositol decreased (2.1 nmol). This could be due to phospholipase A2, in addition to phospholipase C, acting directly on phosphatidylinositol to a greater extent than estimated by accumulation of lysophosphatidylinositol, degraded rapidly by lysophospholipase. Thus, upon high-dose thrombin stimulation of human platelets inositide metabolism via phospholipase C directs initial formation of intracellular second messengers, and sequentially, or in parallel, arachidonate release by phospholipase A2 supplies the larger proportion of arachidonate for syntheses of eicosanoids involved in intercellular communication.