The purpose of this study was to determine whether the synthesis of endogenous nitric oxide (NO) is involved in the apoptosis of murine L929 transformed fibroblasts. L929 parental cells and L929 cells selected for resistance to tumor necrosis factor (TNF-alpha) were incubated in vitro with various concentrations of TNF-alpha, interleukin-1, and lipopolysaccharide (LPS) in the presence or absence of mouse interferon-gamma (IFN-gamma). The combination of subthreshold concentrations of IFN-gamma with the cytokines or LPS produced significant cell death within 48 h incubation. This cell death was associated with the induction of high levels of NO. Both cell death and NO production were significantly inhibited by the addition of N(G)-methyl-L-arginine (NMA), a specific inhibitor of nitric oxide synthase. The synergistic cytotoxicity was associated with extensive internucleosomal DNA fragmentation. NMA also inhibited this process. These data demonstrate the involvement of endogenous NO in cytokine-induced apoptosis of transformed cells.