The nitric oxide (NO) donors S-nitrosopenicillamine or DetaNONOate, which release NO at a rate of 0-15 nM sec-1, were exposed to rat aortic vascular smooth muscle cells for a period of 0-24 h. This treatment resulted in an increase in total glutathione levels of two- to threefold under conditions where no cytotoxicity was detected. The signaling pathways do not involve activation of protein kinase G Iα nor are they cGMP dependent. Oxidation of reduced glutathione (GSH) was found after exposure to NO for 3- 4 h at rates of formation at or above 8 nM sec-1. Increased intracellular GSH was due to enhanced expression of the rate-limiting enzyme for GSH synthesis, γ-glutamylcysteine synthetase. Since NO has been shown previously to protect cells against oxidative stress, we propose that the increase in GSH by NO is a potential mechanism for enhancing the antioxidant defenses of the cell. This result also has important implications for identifying redox- sensitive cell signaling pathways that can be activated by NO.