Environmental factors, such as viral infection, have been implicated in the destruction of β-cells during the development of autoimmune diabetes. Double-stranded RNA (dsRNA), produced during viral replication, is an active component of a viral infection that stimulates antiviral responses in infected cells. Previous studies have shown that treatment of rat islets with dsRNA in combination with υ-interferon (IFN-υ) results in a nitric oxide-dependent inhibition of glucose-stimulated insulin secretion. This study examines the role of nuclear factor-κB (NF-κB) and the dsRNA-dependent protein kinase (PKR) in dsRNA + IFN-υ-induced nitric oxide synthase (iNOS) expression and nitric oxide production by rat, mouse, and human islets. Treatment of rat and human islets with dsRNA in the form of polyinosinic-polycytidylic acid (poly IC) and IFN-υ resulted in iNOS expression and nitric oxide production. Inhibitors of NF-κB activation - the proteasome inhibitor MG-132 and the antioxidant pyrrolidine-dithiocarbamate (PDTC) - prevented poly IC + IFN-υ-induced iNOS expression and nitric oxide production. Incubation of rat islets for 3 h or human islets for 2 h with poly IC alone or poly IC + IFN-υ resulted in NF-κB nuclear translocation and degradation of the NF-κB inhibitor protein, IκB, events that are prevented by MG-132. PKR has been shown to participate in dsRNA-induced NF-κB activation in a number of cell types, including mouse embryonic fibroblasts. However, poly IC stimulated NF-κB nuclear translocation and IκB degradation to similar levels in islets isolated from mice devoid of PKR (PKR-/-) and wild-type mice (PKR+/+). Furthermore, the genetic absence of PKR did not affect dsRNA + IFN-υ-induced iNOS expression, nitric oxide production, or the inhibitory actions of these agents on glucose-stimulated insulin secretion. These results suggest that 1) NF-B activation is required for dsRNA + IFN-υ-induced iNOS expression, 2) PKR is not required for either dsRNA-induced NF-κB activation or dsRNA + IFN-υ-induced iNOS expression by islets, and 3) PKR is not required for dsRNA + IFN-υ-induced inhibition of glucose-stimulated insulin secretion by islets.