The cytotoxic mechanism of action of tumor necrosis factor (TNF) was examined using murine L929 fibrosarcoma cells in vitro. Two cell lines were evaluated: parental TNF sensitive (L929S) (50% cytotoxic concentration, 2-6 ng/ml); and TNF resistant (L929R) (50% cytotoxic concentration, greater than 10,000 ng/ml). The latter resistant cell line was developed by serial passage in increasing concentrations of recombinant human TNF. Sensitive cells demonstrated cytolytic and cytostatic effects at TNF concentrations between 2 and 6 ng/ml, respectively. However, TNF failed to show any selective depression of RNA, DNA, or protein synthesis or ATP content in these cells until general cell death was apparent, as defined by the cell rounding and lifting off the plastic surface. The cytokine also failed to cause DNA single-strand breaks, as detected by alkaline elution techniques. TNF was also found to be no more active in glutathione-depleted cells than in target cells containing normal glutathione levels. In contrast, various nonspecific lysosomotropic agents such as ammonium chloride and D-saccharic acid lactone led to a marked inhibition of the cytotoxic action of TNF in vitro. Furthermore, significant differences in lysosomal enzyme activity were noted between L929S and L929R cells. The changes in L929R cells involved a 50% reduction in total lysosomal protein levels and a marked depression of beta-glucuronidase activity. In contrast, L929R lysosomal hexosaminidase activity was significantly elevated over the L929S cells. From these studies it is concluded that the antitumor activity of TNF does not involve specific inhibition of macromolecular synthesis, ATP production, or the level of reduced thiols. Instead, TNF cytotoxicity appears to require functional lysosomes, which are altered when TNF resistance develops in vitro.