We established an in vitro model to study the influence of ifosfamide treatment on intracellular glutathione (GSH) levels in activated human effector cells with specific phenotypes and immunologic functions. Besides its role as the major intracellular reductant, GSH has been shown to affect the initiation and progression of lymphocyte activation after stimulation with lectins. An incubation of activated human peripheral blood lymphocytes (PBL) with 4-hydroxyifosfamide, the activated form of ifosfamide (4-OH-IF), resulted in a depletion of the intracellular GSH levels and a significant inhibition of the proliferative capacity in a dose-dependent manner. The cytotoxic activity of separated CD3- natural killer (NK) cells and CD3+ allospecific, cytotoxic T lymphocytes (CTL), either untreated or treated with 4-OH-IF at different concentrations, was compared in a standard 51chromium release assay (CML). There were three major findings. (1) The capacity of CD3+ major histocompatibility complex (MHC)-restricted CTL to lyse their specific allogeneic target cells was substantially reduced by preincubation of the effector cells with 4-OH-IF. This inhibition of the lytic activity in CD3+ CTL correlated with a substantial depletion of the intracellular GSH levels in this population. Rapid reconstitution of depleted GSH levels and restoration of cytotoxic activity of CTL was achieved by incubation of the effector cells with thiols, eg, glutathione ester (GSH-ester) or 2-mercaptoethanesulfonate (mesna). (2) In contrast, the lytic activity in CD3- NK cells was not substantially affected (up to 100 mumol/L 4-OH-IF). This result correlates with the capacity of NK cells to maintain their intracellular GSH levels after an ifosfamide treatment. (3) In comparison with CD3+ CTL, CD3- NK cells are more resistant to an ifosfamide treatment because they have higher initial GSH levels and a more than fourfold higher relative rate of GSH synthesis.