Tumor necrosis factor (TNF) is selectively cytotoxic for some tumor cells in vivo and in vitro. We determined whether TNF-mediated cytotoxicity for TNF-sensitive tumor targets was related to TNF-stimulated production of NO by the tumor cell itself. We found that a cell line that was sensitive to TNF-mediated cytotoxicity produced NO in response to TNF as measured by the accumulation of nitrite in the supernatants of TNF-stimulated cells. Production of NO in response to TNF was inhibited by the competitive substrate inhibitor, NG-monomethyl-L-arginine (NMMA). The kinetics of NO production in response to TNF indicated that most of the NO was produced during the first 24 h and peaked after 48 h of culture and that TNF-stimulated NO production was dose dependent. TNF-resistant cell lines produced less NO than a TNF-sensitive cell line, and the amount of nitrite produced correlated with the relative sensitivity of each cell line to TNF-mediated cytotoxicity. In addition, recombinant interferon-gamma augmented the amount of NO produced in response to TNF by both sensitive and resistant cells and correspondingly enhanced the susceptibility of resistant cells to TNF cytotoxicity. Both sensitive and resistant cells were sensitive to NO, however, in that NO generated exogenously by culture in the presence of sodium nitroprusside was cytotoxic for both sensitive and resistant cells in a dose-dependent manner. We were unable, however, to demonstrate directly a role for NO in TNF-mediated cytotoxicity as NMMA- and arginine-free media provided little protection from TNF-mediated cytotoxicity. We tentatively conclude that the ability of adherent murine tumor cells to produce nitric oxide in response to TNF correlates directly with their level of sensitivity to TNF-mediated cytotoxicity, although NO thus produced appears not to be directly involved in the cytotoxic mechanism.