The addition of 0.1 muM ionophore A23187 to washed platelets incubated in citrated saline caused massive release of stored serotonin accompanied by intracellular accumulation of inosine monophosphate, but produced no detectable influx of externally added calcium or abnormal structural alterations. With increasing ionophore concentration there was a significant influx of calcium and a drastic alteration in the platelet ultrastructure. The increase in ionophore concentration was accompanied by the conversion of the major part of metabolic adenine nucleotides to inosine monophosphate and an almost complete blockage of further conversion to inosine and hypoxanthine. The metabolic changes were accentuated by the addition of calcium at concentrations less than 1/10 of the citrate concentration. In the presence of Ca++, or when citrate was omitted, there was a substantial leakage of cytoplasmic material, which at times suggested complete exchangeability between cytoplasm and extracellular medium. Our findings are consistent with the hypothesis that the platelet release reaction is triggered by intracellularly bound calcium. They also suggest that the application of high ionophore concentration has a toxicologic rather than a physiologic effect on platelets, and that a weak chelator added during incubation with the ionophore can in the absence of divalent cations prevent cell destruction, but not the toxic effect on cell metabolism.