Fas antigen is a member of the tumor necrosis factor receptor family that transduces a lethal signal to the Fas-sensitive cells. We previously established the Fas-resistant variant cell lines LAC2D1R and JKT2D1R from the parental Fas-sensitive cell lines, SUPT13 and Jurkat, respectively. Recently, we isolated the Fas-resistant variant CEM2D1R from CCRF-CEM. All of the variants were Fas+ but resistant to Fas-mediated apoptosis. Further biochemical analysis revealed that the intracellular glutathione (GSH) content of the Fas-resistant variants was higher than in the original cells. When the Fas-resistant variants were incubated with buthionine sulfoximine (BSO) or in GSH-free/cysteine-free medium to deplete GSH, Fas resistance was reversed. Incubation of the cells with cycloheximide also decreased intracellular GSH and reversed the Fas resistance. Furthermore, incubation of activated peripheral blood lymphocytes with BSO enhanced Fas-mediated apoptosis. When the Fas-sensitive cells were incubated with N-acetylcysteine (NAC), intracellular GSH was increased and Fas-mediated apoptosis was blocked. In contrast, Fas-resistant variants, as well as Fas-sensitive cells pre-treated with NAC remained susceptible to allogeneic lymphokine-activated killer cells, most likely due to perforin-dependent killing. The results suggest that Fas-mediated apoptosis, but not perforin-dependent killing, is modulated by intracellular GSH in human T lymphocytes.