The mechanism whereby tumor necrosis factor (TNF) kills mammalian cells is not well understood, although oxidative damage has been suggested by several investigators. Further, it is not known why cells vary in their responsiveness to TNF. We show that the cytotoxic effect of TNF toward TNF-sensitive L929 cells is blocked under hypoxic conditions, suggesting a critical role of molecular oxygen and reactive oxygen species. To test whether cellular resistance to reactive oxygen species could provide resistance to TNF, we derived a variant strain from L929 cells by chronic exposure to an oxidizing agent, hydrogen peroxide (H2O2). These cells exhibit marked resistance to TNF as well as to H2O2. This cross-protection provides additional evidence that mechanisms of resistance to oxidative damage are causally related to TNF-induced cell death. Scatchard analysis of TNF binding did not reveal significant differences between the H2O2-resistant line and the wild-type L929 line. On the other hand, analyses of antioxidant enzymes and glutathione levels in cells of the wild-type and the H2O2-resistant lines revealed several potentially important differences. Before exposure to TNF, the H2O2-resistant variants have elevated catalase activity, decreased activity of total glutathione-S-transferase (GST), and similar superoxide dismutase (SOD) activities. Exposure to TNF led to alteration in CuZnSOD activity, and much more so in the variants than in the wild-type L929 cells. However, no significant change in MnSOD activities in cells of either cell line was observed. Total GST activity was not altered appreciably by TNF in either cell line, but Western analysis showed that the level of alpha GST isozyme was increased and mu GST isozyme decreased in the H2O2-resistant variants. Furthermore, alterations in total glutathione content were observed in both the control and the variant cells.