The ability of T cell-derived cytokines to induce the expression of the nitric oxide synthase (NOS) gene in murine peritoneal macrophages was examined. IL-2 or TNF-alpha alone had no effect either on gene expression or enzyme activity, whereas IFN-gamma had only modest activity. When IL-2 or TNF-alpha were used in combination with IFN-gamma, there was a marked cooperative induction of both mRNA and enzyme activity. The cooperative effects were truly synergistic, as the consequences of combined cytokine treatment were many times greater than was seen with any of the agents acting independently. The expression of NOS mRNA and enzyme activity in response to combined lymphokine treatments was a continuous process reaching optimal levels between 24 and 48 h after stimulation. Concentration dependency for both IL-2 and TNF-alpha suggested that their effects were mediated through interaction with the corresponding defined cell surface receptors. Human rTNF-alpha was as effective a stimulus as murine TNF-alpha; because human TNF-alpha is recognized only by the p55 Type II TNF receptor, this structure appears to mediate the response to TNF-alpha. When IL-2 and TNF-alpha were added at saturating doses in the presence of IFN-gamma, there was an additive effect on NOS mRNA expression suggesting that IL-2 and TNF-alpha cooperate with IFN-gamma through at least partially distinct intracellular signaling pathways. Expression of NOS mRNA in response to IFN-gamma/IL-2 or IFN-gamma/TNF-alpha treatment required protein synthesis, suggesting that cooperative cytokine induction of NOS involves the intermediate expression of new gene products. Such molecular controls for regulation of inducible macrophage gene expression can be contrasted with regulatory control of other inflammatory genes such as IP-10 and TNF-alpha.