Vascular endothelial cells synthesize nitric oxide (NO) in response to agonists that elevate cytosolic free Ca2+ concentrations. Once activated, NO synthase (NOS) requires arginine, NADPH, and O2 as cosubstrates. In this study, we investigated the role of O2 in regulating endothelial NOS activity in intact bovine aortic endothelial cells by measuring the rate of nitrite (NO2-) and nitrate (NO3-) production after conversion of NO2- to S-nitrosoglutathione before analysis or after reduction of NO2- and NO3- to NO using acidic vanadium chloride. The basal rate of NO2- production was 6.5 +/- 0.8 pmol.min-1.mg protein-1. Thapsigargin (TG, 1 microM) elevated free cytosolic Ca2+ concentration and increased the rate of NO2- synthesis. At maximal concentrations of TG, the rate of stimulated NO2- production was linear for at least 20 min and was eightfold higher than the basal rate (53.5 +/- 1.8 pmol.min-1.mg protein-1). Incubation of cells in gas mixtures chosen to produce PO2 values in the physiological range led to a progressive fall in the rate of TG-stimulated NO2- production, as O2 concentrations were reduced from that of room air. The half-maximal effective concentration for NO2- production by intact cells was found to occur at 38 Torr. PO2 values higher than that of room air did not lead to a change in the rate of TG-stimulated NO2- production. To confirm that measurement of NO2- accurately reflects total NO production, both NO2- plus NO3- were measured in buffer samples from cells incubated in either room air or N2. The sum of these NO oxidation products was inhibited similarly by hypoxia. These findings suggest that O2 is an important determinant of NOS activity in hypoxic tissues or in vascular beds such as the pulmonary arterial or fetal circulation where PO2 values in the range of 40 Torr are encountered normally.