Statins are 3-hydroxyl-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors used for the treatment of hypercholesterolemia. It was recently reported that statins inhibit in vitro hepatitis C virus (HCV) RNA replication. We here report that, of five statins studied, mevastatin and simvastatin exhibit the strongest in vitro anti-HCV activity, lovastatin and fluvastatin have moderate inhibitory effects, and pravastatin is devoid of an antiviral effect. A combination of statins with interferon-alpha (IFN-α) or HCV nonstructural (NS)5B polymerase or NS3 protease inhibitors results in an additive antiviral activity in short-term (3 days) antiviral assays. Neither statins, at a concentration of five-fold their median effective concentration (EC50) value, nor polymerase, protease inhibitors, or IFN-α, at concentrations 10- or 20-fold their EC50 value, were able to clear cells from their replicon following four or six consecutive passages of antiviral pressure. However, the combination of HCV polymerase or protease inhibitors with mevastatin or simvastatin resulted in an efficient clearance of the cultures from their replicon. In colony formation experiments, mevastatin reduced the frequency or prevented the selection of HCV replicons resistant to the nonnucleoside inhibitor HCV-796. Conclusion: A combination of specific HCV inhibitors with statins may result in a more profound antiviral effect and may delay or prevent the development of resistance to such inhibitors. (HEPATOLOGY 2009.)