The endoperoxide prostaglandin G2 (PGG2) induced platelet aggregation as well as the platelet release reaction (release of ADP and serotonin) when added to human platelet-rich plasma. Formation of a metabolite of PGG2 [8-(l-hydroxy-3-oxopropyl)-9,12L-dihydroxy-5,10-heptadecadienoic acid] and a lipoxygenase product [12L-hydroxy-5,8,10,14-eicosatetraenoic acid] accompanied the release reaction caused by aggregating agents such as collagen, ADP, epinephrine, and thrombin. Indomethacin inhibited the release reaction and PGG2 formation induced by these agents but had no effect on PGG2-induced release reaction. The aggregating effect of PGG2 was abolished by furosemide, which is a competitive inhibitor of ADP-induced primary aggregation. These data indicate that the aggregating effect of PGG2 is due to release of ADP and that PGG2 synthesis is required for induction of the release reaction by various aggregating agents. A subject with a hemostatic defect due to abnormal release mechanism [decreased aggregation with epinephrine (second wave) and collagen and normal platelet ADP] had a deficiency of the cyclo-oxygenase that catalyzes formation of PGG2. Normal aggregation and release reaction were obtained with added PGG2. Ii is concluded that the endoperoxide (PGG2) is essential in normal hemostasis because of its role in initiating the release reaction required for aggregation by collagen and the second wave of aggregation caused by, e.g., ADP.