Hepatitis C virus (HCV) persists in the majority of infected individuals and is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Chronic hepatitis C is currently treated with interferon (IFN)-α or with a combination of IFN-α and ribavirin. The availability of an HCV replicon system (Lohmann et al., Science 285, 110-113, 1999) allowed the investigation of the effects of IFN on genuine HCV replication in cultured cells. It is shown here that IFN-α inhibits subgenomic HCV RNA replication in HuH-7 human hepatoma cells. Immunofluorescence, Western blot and Northern blot analysis revealed that levels of both HCV protein and replicon RNA were reduced after treatment with IFN-α in a dose-dependent manner. In further experiments, it was investigated whether MxA plays a role in the inhibition of HCV. The human MxA protein is an IFN-induced GTPase that has antiviral activity against various RNA viruses. However, HCV RNA replication was not affected in transfected HuH-7 cells that transiently overexpressed MxA. Moreover, a dominant-negative mutant of MxA did not interfere with the antiviral activity of IFN-α against HCV RNA replication. Taken together, these results demonstrate that IFN-α inhibits HCV replicons via an MxA-independent pathway.