Activity of both nitric oxide (NO) synthase (NOS) and cyclooxygenase (COX) plays an important role in the regulation of platelet function. NO has been shown to directly activate COX. This study was designed to determine whether products of the COX pathway in turn regulate NOS activity. Human platelets were incubated with aspirin, indomethacin, the selective thromboxane A₂ synthase inhibitor U-63557A, or the prostaglandin H₂-thromboxane A₂-receptor blocker SQ-29548 for 1 h at 37°C. Multiple indexes of the activity of the L-arginine-NO pathway and changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_i) were measured in platelets. Both aspirin and indomethacin decreased NOS activity, measured as the conversion of L-arginine to L-citrulline and nitrite (+nitrate) formation, in platelets in a concentration-dependent fashion. Aspirin also decreased guanosine 3',5'-cyclic monophosphate accumulation in platelets. The NOS inhibitory effects of these aspirin and indomethacin effects were reversed by coincubation with the thromboxane A₂ analog U-46619 or an excess of CaCl₂. Incubation of COX inhibitors with platelets was associated with significant reductions in basal as well as thrombin-stimulated [Ca²⁺]_i, and the reduction in [Ca²⁺]_i was reversed by U-46619. Incubation of platelets with U-63557A and SQ-29548 resulted in inhibitory effects on NOS activity qualitatively similar to those of COX inhibitors. The effects of COX inhibitors or U-63557A were not associated with a change in NOS protein expression in platelets. These data suggest that NOS activity in human platelets is inhibited by COX inhibitors, mediated, at least in part, via suppression of thromboxane A₂ and [Ca²⁺]_i mobilization in platelets.