Environmental factors, such as viral infection, have been implicated as potential triggering events leading to the initial destruction of pancreatic β cells during the development of autoimmune diabetes. Double-stranded RNA (dsRNA), the active component of a viral infection that stimulates antiviral responses in infected cells, has been shown in combination with interferon-γ(IFN-γ) to stimulate inducible nitric oxide synthase (iNOS) expression and nitric oxide production and to inhibit β cell function. Interferon regulatory factor-1 (IRF-1), the activation of which is induced by dsRNA, viral infection, and IFN-γ, regulates the expression of many antiviral proteins, including PKR, type I IFN, and iNOS. In this study, we show that IRF-1 is not required for dsRNA + IFN-γ-stimulated iNOS expression and nitric oxide production by mouse islets. In contrast to islets, dsRNA + IFN-γ fails to induce iNOS expression or nitric oxide production by macrophages isolated from IRF-1-/- mice; however, dsRNA + IFN-γ induces similar levels of IL-1 release by macrophages isolated from both IRF-1-/- and IRF-1+/+ mice. Importantly, we show that dsRNA- or dsRNA + IFN-γ-stimulated IRF-1 expression by mouse islets and peritoneal macrophages is independent of PKR. These results indicate that IRF-1 is required for dsRNA + IFN-γ-induced iNOS expression and nitric oxide production by mouse peritoneal macrophages but not by mouse islets. These findings suggest that dsRNA + IFN-γ stimulates iNOS expression by two distinct PKR-independent mechanisms; one that is IRF-1-dependent in macrophages and another that is IRF-1-independent in islets.