Nitric oxide synthases (NOS) require NADPH and tetrahydrobiopterin (H4biopterin) to convert L-arginine to L-citrulline. The additional requirement and effects of thiols during purification and activity assays of NOS are unclear; for example, glutathione (GSH) has been reported to stimulate or, in the presence of catalase, to inhibit enzyme activity. We therefore studied the effects of different thiols, thiol reagents, antioxidants, and H4biopterin-regenerating systems on purified porcine cerebellum NOS. GSH in the presence of catalase did not inhibit NOS. In contrast, GSH and, to a lesser degree, several other thiols consistently stimulated total L-arginine turnover up to 4-fold. In the presence of GSH, Vmax of NOS was increased, the usually observed loss of activity during the 15 min assay was less dramatic, and the apparent S0.5 value for H4biopterin decreased. Stabilization of NOS activity by GSH was augmented by protein disulfide isomerase (PDI), indicating that, at least in part, GSH acted by reductive protection of NOS protein thiols. Consistent with this, four different protein thiol reagents abolished NOS activity. In other experiments, specific allosteric binding was excluded as a potential mechanism of GSH regulation of NOS. In addition, GSH may affect NOS kinetics by recycling or preventing the autoxidation of H4biopterin. In support of this, the non-thiol reductant ascorbate and dihydropteridine reductase mimicked the effects of GSH on NOS kinetics, but not on NOS stability. Thus, NOS activity depends on both H4biopterin and the reduced state of essential protein thiols.