Excessive production of nitric oxide (NO) by inflammatory cells has been implicated in the pathophysiology of multiple sclerosis and EAE. One of major the consequences of nitrosative stress is the formation of protein S-nitrosothiols (RSNOs). Despite numerous studies there is stilI uncertainty as to process by which NO accumulation causes protein S-nitrosylation in intact cells. This work compares the ability of chemically different NO donors to induce protein RSNOs in mt spinal cord (SC) slices. Incubation with ImM S-nitroso-N-acetylpenicilIamine or S-nitrosoglutathione (GSNO) caused a continuous increase in the amount of protein RSNOs reaching a maximum after 2 h. At steady state, ~8% of total protein thiols were nitrosylated with either donor, and comparable electrophoretic patterns of altered proteins were observed. Quanti­tatively similar findings were obtained using SC homogenate, suggesting that the mte of entry of these putative nonpermeable donors into cells is not a limiting factor in RSNO formation. This idea is supported by the fact that the kinetics of protein S-nitrosylation in SC slices incubated with GSNO was identical to that attained with the membrane permeable analog GSNO ethyl ester. Neither the N203-scavenger azide nor the NO-scavenger rutin affected GSNO-induced production of RSNOs, indicating that the reaction occurs by S-transnitrosylation. Moreover, incuba­tion of SC slices with various N-NO, O-NO and C-NO compounds, as well as with the nonpermeable NO donor spermine NONOate, failed to induce protein S-nitrosylation. Together these results suggest that there is a restricted set of protein thiols that can be nitrosylated and that GSNO could be an effective physiological NO-donor if accumulated outside the cells. Supported by NIH grant NS 47448.