The aim of the present study was to test the hypothesis that exposure of astrocytes depleted of glutathione (GSH) to simulated ischemia conditions in vitro and treated with immunosuppressant immunophilin ligands (cyclosporin A (CsA) and FK506) can increase intracellular GSH levels and that such mechanism may be responsible, at least in part, for their protective effects. In addition, we also compared the antioxidant properties of these immunosuppressants with N-acetylcysteine (NAC), a precursor of GSH synthesis. GSH depletion was induced by 24 h pretreatment with L-buthionine sulfoximine (BSO). Cultures of rat astrocytes were exposed to CsA (1-50 microM) and FK506 (1-1000 nM) and NAC (100 or 200 microM). We examined the effects of these compounds on apoptosis, cell viability, reactive oxygen species production and GSH content. Our study demonstrated that toxicity of simulated ischemia conditions were enhanced when intracellular GSH was depleted, and immunosuppressants (especially 100 nM FK506 and 10 microM CsA) effectively prevented ischemia toxicity in GSH depleted astrocytes. In addition, we have shown that interfering with the generation of GSH and attenuation, the rise of oxidative stress level by NAC may be a powerful tool for prevention of ischemia-induced glial cell damage.