Protein S-nitrosylation has been implicated in the pathophysiology of CNS disorders characterized by extensive nitrosative stress, including multiple sclerosis and its animal model experimental allergic encephalomyelitis. Using rat spinal cord slices we have established that S-nitrosoglutathione (GSNO) is a viable intercellular S-nitrosating agent and that there is a restricted number of proteins available for S-nitrosylation. Moreover, generation of protein nitrosothiols (PrSNOs) with GSNO occurs exclusively via a S-transnitrosation mechanism. To determine the biochemical stability of PrSNOs and the factors involved in the cleavage of S-NO linkage in intact cells, we carried out pulse-chase experiments. Results clearly show that removal of GSNO from the incubation medium leads to a rapid disappearance of PrSNOs (t1/2<1h). When the chase was carried out in the presence of the glutathione depletor diethyl maleate, PrSNOs were biochemically stable indicating that glutathione (GSH) plays a key role in the denitrosylation process. Furthermore, there is an inverse relationship between GSH levels and amount of PrSNOs remaining after the chase. Interestingly, the removal of the NO moiety from PrSNOs was not accompanied by a concomitant increase in the amount of protein thiols, suggesting the occurrence of intramolecular disulfides and/or intermolecular disulfides with GSH (i.e. protein glutathionylation). In summary, GSH (alone or via glutathione-dependent enzymes) is necessary for denitrosylation of CNS protein in intact cells. The identification of the factors that control protein S-nitrosylation/S-denitrosylation in the nervous system is essential for understanding the neuropathological consequences of excessive NO production. Supported by NIH grant NS 47448.