Nitric oxide (NO) synthase is a hemoprotein containing several cysteinyl residues including thiolate as its proximal heme ligand. Exposure to NO is known to induce S-nitrosylation of protein thiols and modulation of enzyme activities, including the catalytic activity of NO synthase. Because S-nitrosylation of vicinal thiols promotes disulfide formation, we determined whether exposure to NO results in modulation of the catalytic activity of NO synthase and whether disulfide reduction catalyzed by thioredoxin/thioredoxin reductase (T/TR) and/or by glutaredoxin restores the catalytic activity of NO synthase in pulmonary artery endothelial cells (PAEC). Exposure of intact PAEC, isolated total membranes, plasma membranes, or purified NO synthase to NO significantly reduced NO synthase catalytic activity. Similarly, exposure of isolated total membranes or purified NO synthase to potassium ferricyanide (FeCN) also reduced catalytic activity of NO synthase in a concentration-dependent fashion. Although the catalytic activity of NO synthase was significantly reduced following exposure of intact cells to NO, the expression of NO synthase mRNA was unchanged. NO synthase activity in intact cells or isolated membranes exposed to nitrate, nitrite, or 10 ppm nitrogen dioxide gas was comparable to controls. Incubation in the presence of oxyhemoglobin prevented but did not reverse NO-induced inhibition of NO synthase. Incubation in the presence of T/TR but not glutaredoxin reversed NO-induced reduction of NO synthase activity and a purified enzyme preparation exposed directly to NO. Similarly, FeCN-induced reduction of NO synthase activity was also reversed in the presence of T/TR but not by glutaredoxin. These results demonstrate that the interaction of NO with the regulatory domain of NO synthase protein is responsible for post-translational reduction of its catalytic activity. Thioredoxin-regulated reversal of NO-induced modulation of NO synthase protein suggests that an oxidative conformational change in vicinal thiols, resulting in the formation of intramolecular or intermolecular disulfides or both, is involved.