Peroxynitrite (ONOO(-)) is critically involved in the pathogenesis of cardiovascular diseases. Reaction with glutathione (GSH) was proposed to be a major detoxification pathway of ONOO(-) in the biological system. This study was undertaken to determine if chemically elevated intracellular GSH affords protection against ONOO(-)-mediated toxicity in vascular cells. Incubation of aortic smooth muscle A10 cells with 3H-1,2-dithiole-3-thione (D3T) led to a concentration- and time-dependent elevation of cellular GSH. Treatment of the cells with D3T also augmented protein and gene expression of gamma-glutamylcysteine ligase. To examine the effects of D3T-induced GSH on ONOO(-)-mediated toxicity, we pretreated A10 cells with D3T and then exposed them to either authentic ONOO(-) or the ONOO(-) generator, 3-morpholinosydnonimine. We observed that D3T pretreatment of A10 cells resulted in a significant protection against ONOO(-) cytotoxicity. Conversely, depletion of cellular GSH by buthionine sulfoximine (BSO) caused a marked potentiation of ONOO(-) cytotoxicity. To further demonstrate the causal involvement of GSH induction in D3T cytoprotection, we cotreated A10 cells with BSO to abolish D3T-induced GSH elevation. BSO cotreatment was found to greatly reverse the protective effects of D3T on ONOO(-)-elicited cytotoxicity. Taken together, our results demonstrate that upregulating GSH biosynthesis by D3T results in a marked protection against ONOO(-)-induced toxicity in vascular cells.