In this study we examined the impact of hepatitis C virus (HCV) RNA replication on the innate antiviral response of the host cell. Replication of an HCV subgenomic replicon stimulated the activation of the interferon (IFN)-beta promoter and the production of IFN in human hepatoma cells. Using a variety of functional assays, we found that HCV RNA replication induced the activation and DNA-binding activity of NFkappaB and interferon regulatory factor (IRF)-1. In addition, microscopy experiments revealed a higher frequency of cells containing the nuclear-localized, active form of IRF-3 in HCV replicon cultures versus control cultures. Consistent with these observations, cells harboring the HCV replicon exhibited high basal level expression of a subset of IFN-stimulated antiviral genes. Our results indicate that HCV RNA replication can stimulate cellular antiviral programs that contribute to the assembly and activation of the IFN-beta enhanceosome complex and initiation of the antiviral state. Stable HCV RNA replication in the face of the host antiviral response suggests that HCV may encode one or more proteins capable of overcoming specific antiviral processes, thereby supporting persistent infection.