Activated interleukin-2 (IL-2)-dependent T cells express high levels of Bcl-2 protein. On cytokine withdrawal, Bcl-2 expression decreases and the cells die rapidly by apoptosis. We have previously shown that the survival of IL-2-deprived T cells can be promoted by factor(s) secreted by fibroblasts. Here we report that reduced glutathione (GSH), but not its oxidized counterpart GSSG, also enhances the in vitro survival of these cells. Exogenous GSH mediates its effect intracellularly, as (1) endogenous glutathione concentrations are increased up to fivefold in the presence of GSH, and (2) acivicin, an inhibitor of transmembrane GSH transport, abrogates GSH-dependent survival. The GSH-rescued T cells do not proliferate and express only low levels of Bcl-2, resembling W138 fibroblast-rescued T cells. We, therefore, investigated a role for GSH in fibroblast-promoted T-cell survival. We show that W138-promoted survival results in elevated GSH levels in surviving T cells and is abrogated by buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. Furthermore, both W138-promoted T-cell survival and GSH upregulation are associated with large molecular weight molecules (>30 kD). Thus, the upregulation of GSH by W138 fibroblasts appears to be crucial in their ability to enhance the survival of cytokine-deprived activated T cells in vitro.