A23187-treated platelets secrete dense granule constituents during centrifugation, an artifact that is presented by prior formalin fixation (Holmsen, H., and Setkowsky-Dangelmaier, C. A. (1977) Biochim. Biophys. Acta 497, 46-61). With this improved assay, A23187 induced no secretion in nonaggregating platelets and maximal secretion in aggregating platelets within 3 min. Further incubation gave a slow, submaximal secretion in nonaggregating cells. Acetylsalicylate abolished secretion in both systems. EDTA, but not ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, strongly enhanced secretion in nonaggregating platelets suspended in Mg2+-containing, Ca2+-free Tyrode's solution. Without added Mg2+, A23187 gave maximal secretion in nonaggregating platelets which was abolished by added Mg2+. Preincubation of A23187 and MgCl2 gave inhibition patterns which clearly suggested that formation of Mg.A23187 species was the cause of inhibition. Ca2+, but not Sr2+, inhibited A23187-induced secretion in the same manner as Mg2+. These findings suggest that the platelet plasma membrane has no or very little permeability for Ca2+ . and Mg2+ . A23187 species. In the physiological suspending medium, Ca2+-free Tyrode's solution, A23187-induced platelet secretion is markedly enhanced by close cell contact (aggregation) and has an absolute requirement for production of prostaglandins and/or thromboxanes. Therefore, the widely held view that secretion is directly triggered by A23187-induced increase in the cytoplasmic Ca2+ concentration is not applicable to platelets.