Recent studies have characterized endothelium-derived relaxing factor (EDRF) as nitric oxide which appears to exert its inhibitory effect on human platelets by elevating intracellular levels of cyclic nucleotides (cyclic AMP, cyclic GMP). In this study we have confirmed nitric oxide to be a potent inhibitor of agonist-induced platelet activation. It effectively blocked thrombin-induced calcium mobilization, and also lowered agonist-elevated calcium levels in Fura-2-loaded platelets. Nitric oxide induced significant elevation of cyclic GMP levels, but did not influence cyclic AMP levels. Elevation of cyclic GMP levels did not result in enhanced phosphorylation of the 49 kDa protein. Furthermore, cell-permeant analogues (dibutyryl cyclic GMP and 8-bromo cyclic GMP, 100 μM) were poor inhibitors of agonist-induced platelet aggregation and calcium mobilization. Inhibitors of cyclic GMP phosphodiesterase (M and B 22948, MY 5445) potentiated the inhibitory action of nitric oxide. However, at the concentrations at which they potentiated, they also were potent inhibitors of platelet function. Although adrenaline has not been implicated as a guanylate cyclase inhibitor, it effectively reversed the effect of nitric oxide on platelet function, even in the presence of cyclic GMP phosphodiesterase inhibitors. In addition to nitric oxide, ascorbic acid, 5-hydroxytryptamine and the prostaglandin endoperoxides also increase cyclic GMP levels in platelets but exert no inhibitory effect on platelet function. The results obtained in this study do not support the concept that nitric oxide exerts its inhibitory effect by elevating cyclic GMP levels. It is a potent inhibitor of platelet function, but the mechanism by which it exerts this effect on platelet activation is not clear.