W e have previously shown that the erythroagglutinating phytohemagglutinin (E-PHA) from Phaseolus 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. We now find 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 of E-PHA per cell, and we estimate 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 75-85% of platelet [¹⁴C]-serotonin, with half-maximal release occurring less than 0.5 min after thrombin addition. E-PHA, however, induces release of only 30-60% of platelet serotonin at a 10-fold slower rate. In addition, utilizing electron microscopy, we have observed striking differences in the morphological 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.