Inhibitors of the endoplasmic reticulum Ca(2+)-ATPase like thapsigargin (TG) and 2,5-di (tert-butyl)-1,4-benzohydroquinone (tBuBHQ) cause increases in cytosolic calcium in intact human platelets resulting from prevention of reuptake. A maximal concentration of TG (0.2 microM) mobilized 100% of sequestered Ca2+ compared to the action of a receptor agonist like thrombin (0.1 U/ml). A maximal dose of tBuBHQ (50 microM) stimulated release of about 40% of intracellular calcium compared to thrombin and TG. The reduced ability of tBuBHQ to release calcium can be explained with an inhibitory effect on the cyclooxygenase pathway (Ki approximately 7 microM). Therefore tBuBHQ is not able to cause platelet aggregation compared to TG. In the presence of a cyclooxygenase inhibitor or a thromboxane A2 receptor antagonist the action of TG is identical to that observed with tBuBHQ. Generally, inhibition of calcium sequestration does not automatically result in platelet activation. In contrast to a receptor mediated activation Ca(2+)-ATPase inhibitors require the self-amplification mechanism of endogenously formed thromboxane A2 to cause a similar response. We conclude that the calcium store sensitive to Ca(2+)-ATPase inhibitors is a subset of the receptor sensitive calcium pool.