Persistent infection with hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. All treatments known so far rely on the antiviral activity of interferon alfa (IFN-alpha) that is given alone or in combination with ribavirin. Unfortunately, only a fraction of the patients clear the virus during therapy and for those who do not respond there is currently no alternative treatment. Selectable subgenomic HCV RNAs (replicons) have been recently used to investigate the effect of IFN-alpha on HCV replication. However, it has not yet been analyzed whether other cytokines also play a role in the innate immune response against HCV. Here we show that IFN-gamma inhibits protein synthesis and RNA replication of subgenomic and genomic HCV replicons. We further show that the inhibitory action of IFN-gamma does not rely on the production of nitric oxide or the depletion of tryptophan. In conclusion, our results suggest that cytotoxic T cells and natural killer cells may contribute to HCV clearance not only by cell killing but also by producing IFN-gamma, thereby enhancing the intracellular inhibition of viral replication.