Our previous studies have demonstrated that platelets possess ATP purinergic receptors in addition to the ADP, P2T, receptor. Occupancy of the P2 receptor by ATP inhibited agonist-induced platelet aggregation. This study demonstrated that the mechanism of inhibition may involve ATP inhibition of agonist-induced mobilization of internal calcium. Within the cardiovascular system, the ATP inhibition of calcium mobilization is unique to platelets. All other cell types in the cardiovascular system, where calcium mobilization is affected by extracellular ATP, responded with an increased mobilization as opposed to inhibition. The platelet inhibitory response to ATP was enhanced by the addition of an ATP generating system, creatine phosphate/phosphocreatine kinase. ATP and ATP analogues were found to inhibit calcium mobilization with a rank order of alpha beta-methylene ATP, beta gamma-methylene ATP approximately ATP > benzoyl ATP > 2 methylthio ATP which is a characteristic of P2x-like receptors. The inhibitory effect of ATP could be abrogated by prolonged treatment of platelets with the P2x desensitizing agent, alpha beta-methylene ATP. Also, UTP and CTP were approximately as effective inhibitors as ATP while GTP was not. ATP competition with ADP for the P2T receptor was excluded in studies with platelets derived from an aspirin-treated individual which were essentially insensitive to ADP. The agonist-induced calcium mobilization and inhibition by ATP occurred with the thromboxane A2 mimetic, U46619, collagen and thrombin; however, the kinetics of mobilization varied somewhat with the different agonists. The responses to extracellular ATP were independent of extracellular Ca2+, where 1 mM calcium or 0.3 mM EGTA was added to the reaction mixture. The inhibition of calcium mobilization coupled to inhibition of platelet aggregation by extracellular ATP may serve an important physiologic role. ATP, released from activated platelets at localized sites of vascular injury, may help to limit the size of the platelet plug-clot that, if left unregulated, could occlude the injured blood vessel.