It was previously shown that the erythroagglutinating phytohemagglutinin (E PHA) from P. vulgaris binds to the surface of intact human platelets and that adenylate cyclase activity in the particulate fraction of E PHA treated platelets is lower than in comparable controls. It is now found that E PHA induces release of [14C]serotonin from platelets. Release follows binding of E PHA, and a haptenic inhibitor of E PHA binding prevents induction of release. E PHA does not produce platelet lysis and has little effect on [14C]serotonin uptake. Platelets possess approximately 300,000 receptor sites for E PHA per cell, and it is estimated that about 15% of these sites must be occupied by E PHA to initiate the release reaction. Prior incubation of platelets with prostaglandin E1, theophylline, or dibutyryl cyclic AMP prevents E PHA induced release, although these agents have little effect on E PHA binding to platelets. Thrombin and E PHA produce different rates and extents of serotonin release. Thrombin (1 U/ml) causes release of 75 to 85% of platelet [14C]serotonin, with half maximal release occurring less than 0.5 min after thrombin addition. E PHA, however, induces release of only 30 to 60% of platelet serotonin at a 10 fold slower rate. In addition, utilizing electron microscopy, striking differences were observed in the morphologic changes that occur in platelets exposed to E PHA as compared with thrombin. Thus, the platelet release reaction may be triggered in part by binding of E PHA to the cell surface, but this reaction only partially resembles that produced by thrombin.