Previous observations that platelet guanosine 3′, 5′-monophosphate (cyclic GMP) accumulation occurred during platelet aggregation and also in the presence of nitroprusside, which inhibits aggregation, prompted an evaluation of the effects of nitroprusside, nitric oxide, and related agents on human platelet aggregation. Adenosine diphosphate (ADP) was employed to induce irreversible aggregation of human platelets in platelet-rich plasma. Nitroprusside, nitric oxide, and N-methyl-N′-nitro-N-nitrosoguanidine, in a concentration-dependent manner, inhibited ADP-induced platelet aggregation, markedly activated soluble platelet guanylate cyclase (70–280-fold), and markedly elevated platelet cyclic GMP levels (100–200-fold). The gaseous phase of cigarette smoke, which contains nitric oxide, also inhibited platelet aggregation and elevated platelet cyclic GMP levels. In addition to attenuating aggregation, these agents were effective in disaggregating platelets, which was associated with a concomitant rise in cyclic GMP levels. Thus, cyclic GMP accumulation was associated with both inhibition and reversal of platelet aggregation. Partial inhibition of platelet aggregation was observed also with the lipophilic 8-bromo derivative of cyclic GMP. Inhibition of platelet aggregation, activation of guanylate cyclase, and elevation of cyclic GMP levels elicited by nitroprusside, nitric oxide, and related agents were partially blocked by methemoglobin, a hemoprotein with a high binding affinity for nitric oxide. These data suggest that nitric oxide may be one common reactive intermediate responsible for guanylate cyclase activation, cyclic GMP accumulation, and inhibition of platelet aggregation elicited by nitroprusside, nitrosuguanidine, and cigarette smoke. Moreover, the observations that platelet cyclic GMP formation is associated with inhibition of platelet aggregation by agents related to nitric oxide indicate that a reevaluation of the role of cyclic GMP in platelet aggregation is warranted.