P latelet aggregation is currently felt to play an important role in the pathogenesis of ischemic vascular disorders. The smooth muscle relaxant, nitroglycerin, has been shown to inhibit platelet aggregation in vitro, but at concentrations that were felt to be unattainable in vivo. Because the in vivo action of nitroglycerin on smooth muscle cells has been shown to depend on the presence of reduced cytosolic sulfhydryl groups, the inhibitory effect of nitroglycerin on platelet aggregation was examined in the presence of the reduced thiol, N-acetylcysteine. Millimolar concentrations of N-acetylcysteine potentiated markedly the inhibitory effect of nitroglycerin on platelet aggregation induced by ADP, epinephrine, collagen, and arachidonate, decreasing the 50% inhibitory concentration (IC50) approximately 50-fold for each of these agents. Other guanylate cyclase activators inhibited ADP-induced aggregation similarly and this inhibition was likewise potentiated by N-acetylcysteine. Platelet guanosine 3',5'-cyclic monophosphate content increased fivefold in the presence of nitroglycerin and N-acetylcysteine 2 min before maximal inhibition of ADP-induced aggregation was achieved, while simultaneously measured cyclic AMP did not change relative to base-line levels. In the absence of N-acetylcysteine, nitroglycerin induced a marked decrease in platelet-reduced glutathione content as S-nitroso-thiol adducts were produced. The synthetic S-nitroso-thiol, S-nitroso-N-acetylcysteine, markedly inhibited platelet aggregation with an IC50 of 6 nM. These data show that N-acetylcysteine markedly potentiates the inhibition of platelet aggregation by nitroglycerin and likely does so by inducing the formation of an S-nitrosothiol adduct(s), which activate guanylate cyclase.