The intracellular redox state is stringently maintained by thiol-based antioxidants to establish a balance for the physiological and physiological roles of reactive oxygen species. The relative contributions of the thioredoxin and glutathione/glutaredoxin systems to intracellular redox balance are incompletely understood, as are the consequences of altered thiol metabolism on eNOS and NO-dependent pathways in the endothelium. We designed duplex siRNA constructs to specifically "knock down" the expression of three key thiol-metabolizing enzymes in cultured aortic endothelial cells. Transfection of siRNA constructs targeting glutathione reductase (GR), cytosolic Trx reductase (TrxR1), or mitochondrial Trx reductase (TrxR2) significantly decreased the intracellular reduced glutathione/oxidized glutathione ratio. siRNA-mediated knockdown of either GR, TrxR1 or TrxR2 markedly suppressed VEGF-induced NO production (measured by an electrochemical NO sensor), and also blocked eNOS enzyme activity (using the [³H]-arginine/[³H]-citrulline assay). Pretreatment of endothelial cells with BCNU, an inhibitor of GR and TrxR, significantly decreased VEGF-induced NO production. siRNA-mediated TrxR2 knockdown led to a marked increase in hydrogen peroxide (H₂O₂) production in endothelial cells. In contrast, knockdown of GR or TrxR1 only slightly increased H2O2 production. Supplementation of endothelial cells with tetrahydrobiopterin prevented the increase in H₂O₂ generation seen with siRNA-mediated knockdown of GR. These studies show that the differential regulation of thiol-metabolizing proteins leads to critical changes in oxidative and nitrosative stress pathways. Greater understanding of the differential regulation of thiol-metabolizing proteins may lead to the development of new pharmacological targets for diseases associated with oxidative stress in the vascular wall.