Tumor necrosis factor-α (TNF) and interferon-γ (IFN) modulate immune responses, as TNF is postulated as a first messenger for priming immune cells and IFN as the second messenger for activation. Nitric oxide (NO) is also an important mediator of immune function, as NO produced by immune cells in response to cytokines such as TNF and IFN may be a cytotoxic end-product effector of immune activation. To examine TNF and IFN modulation of NO production and effects upon allograft survival, we studied the in vitro effect of TNF, IFN, and lipopolysaccharide (LPS, as a nonspecific immune stimulator and TNF promoter) singly or together on NO production in unstimulated rat splenocytes or in response to allogeneic stimulation in MHC mismatch (Brown-Norway, BN, vs Lewis, LEW) mixed lymphocyte reactions. Nitrite as a stable reaction product of NO was determined by a colorimetric method based on the Griess reaction. Interestingly, unstimulated cells had little NO production in response to TNF or IFN; however, following nonspecific (LPS) or allogeneic stimulation there was a significant upregulation of NO production that was synergistically enhanced by the presence of both TNF and IFN. Significantly, anti-TNF antibody inhibited NO production up to 60% in all groups. In vivo studies used a cardiac heterotopic allotransplant model, again BN to LEW. Control recipients received no immunotherapy. Experimental recipients received IFN alone, anti-IFN antibody, anti-TNF antibody, or anti-TNF and anti-IFN antibody. Results from these allograft experiments showed no influence on survival by IFN alone or anti-FN antibody alone. However, anti-TNF antibody extended allograft survival, which was synergistically and significantly enhanced with the addition of anti-IFN antibody. These results demonstrate that cytokines synergistically modulate immune response via NO synthesis, as NO is markedly increased by IFN in the presence of allogeneic stimulation or TNF/LPS. Furthermore, as anti-IFN was only beneficial to allograft survival in the presence of anti-TNF, the priming and activating roles of these cytokines in modulating the alloimmune response via NO production suggest that transplant rejection may be mediated through a distinct cascade. Modulation of cytokine-NO cascades may lead to new therapies.