IL-2 therapy can induce marked oxidative stress via reactive oxygen and nitrogen intermediates. Glutathione, the major intracellular reductant, may become rate limiting to cytotoxic lymphocyte activation and proliferation under these circumstances. N-Acetyl cysteine (NAc-cys) was used to increase intracellular glutathione levels during lymphokine-activated killer (LAK) cell activation by IL-2. Incubation of splenocytes with NAc-cys (0.6 to 1.0 mM) resulted in significant changes in intracellular reduced and total glutathione (92% and 58% increase, respectively) at 96 h. These levels correlated with markedly enhanced cell proliferation (threefold) and cytolytic effector cell generation (> fivefold increase in LU/10(6) cells) induced by the combination of NAc-cys with IL-2. IL-2 exposure by itself unexpectedly increased intracellular reduced glutathione by 43%. IL-2 and NAc-cys were synergistic in increasing glutathione levels (reduced glutathione: 292% increase; total: 251% increase). Inhibition of glutathione synthesis, using L-buthionine-(S,R)-sulfoximine reversed the effects of NAc-cys on intracellular glutathione, as well as cellular proliferation and cytotoxicity. This experiment established that the effects of NAc-cys required de novo glutathione synthesis. In conjunction with IL-2/LAK treatment, oral NAc-cys administration (260 to 900 mg/kg/day for 7 days) significantly decreased tumor progression in a refractory s.c. tumor model. A small fraction of mice (11 to 17%) had complete tumor regressions. NAc-cys may be useful as an adjunct to increase the antitumor activity of IL-2/LAK therapy.