The treatment of human embryonic kidney 293 cells harboring a hepatitis C virus (HCV) subgenomic replicon with the double-stranded RNA (dsRNA) mimic poly(I . C) inhibits HCV RNA replication through an undefined mechanism. Interferon regulatory factor 3 (IRF 3) has been widely postulated to mediate various antiviral responses, and its role in mediating the response to dsRNA in 293 cells was examined. Treating the cells with dsRNA did not induce IRF-3 activation, as measured by nuclear localization or the induction of reporter genes. Moreover, the expression of a dominant negative form of IRF-3 did not affect either colony formation upon transfection of subgenomic replicon RNA or the inhibition of the HCV replicon by dsRNA. Our results suggest that the inhibition of HCV RNA replication by poly(I . C) in 293 cells is independent of IRF-3 activation.