We investigated whether hypoxia (2% O2, approximately 14 mm Hg partial pressure) in comparison to O2 atmospheric pressure (20.9% O2, approximately 140 mm Hg) can affect the cytotoxic effects of tumor necrosis factor-alpha (TNF) on the murine cell line L929. Under hypoxic conditions, L929 cells were significantly less inhibited by TNF treatment, even in the presence of actinomycin D. Moreover, under hypoxic conditions, TNF cytotoxicity was significantly inhibited by glutathione, which has been shown to protect cells against oxidative damage induced by various agents. On the other hand, under aerobic conditions treatment with other antioxidant agents and active species oxygen scavengers, as superoxide dismutase and catalase, did not markedly affect the cytotoxicity of TNF. Since hypoxia occurs normally in most solid tumors, these results are interesting because they suggest a disadvantageous inhibition of the cytotoxic effects of TNF in vivo in hypoxic tissues and confirm that oxygen-dependent metabolic processes or free radicals are required to exert TNF-induced cytotoxicity.