Thrombin-induced platelet aggregation is associated with an increase in intracellular calcium. Epinephrine provokes aggregation in the absence of a rise in intracellular calcium. Adenosine has been postulated as an endogenous inhibitor of platelet aggregation. In this study, the authors examine the effect of adenosine on the rise in intracellular calcium and on platelet aggregation, and the role of cyclic AMP (cAMP) in these actions. Human platelets were obtained from citrated plasma containing 5 micrograms/mL of indomethacin. Intracellular calcium was determined by fura-2 fluorescent dye. Adenosine inhibited thrombin-induced platelet aggregation and the rise in intracellular calcium in a dose-dependent manner. At a concentration of 100 mumol/L, adenosine completely inhibited thrombin-induced aggregation, but only partly inhibited the rise in intracellular calcium (55%). Adenosine also partially inhibited the rise in calcium produced by thrombin in both calcium-containing and calcium-free media, suggesting that adenosine inhibits both calcium influx and calcium mobilization. The effects of adenosine on intracellular calcium, as in the case of platelet aggregation, appear to be linked to adenylate cyclase, since they were prevented by the adenylate cyclase inhibitor 2',5'-dideoxyadenosine (1-mmol/L) and were potentiated by phospho-diesterase inhibition with papaverine (1 mumol/L). Adenosine and dibutyryl-cAMP also inhibited epinephrine-stimulated platelet aggregation in a dose-dependent manner. Thus, it appears that adenosine may inhibit platelet aggregation independently of its ability to decrease cytosolic free calcium.