The dose-dependent induction of platelet aggregation, dense granule secretion and thromboxane formation by the divalent cation ionophores A23187 and ionomycin were compared in citrated platelet rich plasma (PRP), and measured simultaneously with the increases in cytosolic Ca2+levels in aequorin-loaded gel-filtered platelet (GFP). In citrated PRP, both ionophores induced similar extents of aggregation at comparable concentrations, whereas A23187 induced somewhat greater extents of secretion, and substantially greater extents of thromboxane B2(TxB2) formation, than ionomycin. When 5 mM EDTA or EGTA was added to PRP secretion and TxB2formation occurred only with A23187; ionomycin was inactive at all concentrations tested, up to 100 pM. In aequorin-loaded GFP containing 1 mM Ca2+, 1 mM EGTA or 2 mM EDTA, ionomycin as well as A23187 induced these platelet responses, but the concentration dose-response curve for ionomycin was shifted to the right by approximately one order of magnitude relative to that for A23187. Simultaneous measurements of the cytosolic Ca2+([Ca2+]1) increases induced by the two ionophores showed that the increases produced by ionomycin were consistently 2040% less than those induced by A23187 at all ionophore concentrations tested. Analysis of the extents of secretion and TxB, formation obtained in EDTA- or EGTA-containing systems as a function of the [Ca2+]1increases suggested that the data for both ionophores were described by the same [Ca2+]1dose-response curve, indicating that the decreased extents of these responses seen with ionomycin vs A23187 were due primarily, if not solely, to the lower [Ca2+], increases produced by ionomycin. Since ionomycin is theoretically capable of transporting twice as much divalent cation as A23187, these findings in platelets, together with similar findings in certain other cell systems, provide evidence that factors associated with the intracellular environment may differentially affect the abilities of A23187 and ionomycin to induce cellular responses and, more specifically, to release intracellular Ca2+stores.