The recent finding that ribavirin has a mutagenic capacity in a poliovirus replicon model pushing the virus into error catastrophe, provides a possible explanation for the remarkable synergistic effect of ribavirin when combined with interferon in the treatment of chronic hepatitis C virus (HCV)-infected patients. However, ribavirin-induced hypermutation resulting in loss of vital genetic information and viral clearance, does not occur during treatment of HCV-infected patients, as can be inferred from the lack of viral inhibition when treating HCV-infected patients with ribavirin alone. We therefore hypothesized that ribavirin induces mutations in the C-terminal part of the viral NS5A gene, a region found to be correlated with interferon sensitivity. Ribavirin-induced mutations resulting in the appearance of viral variants more sensitive to interferon would explain the synergistic effect of ribavirin when combined with interferon. To test this hypothesis we retrospectively analysed sequences of the C-terminal half of the NS5A gene before and during treatment in six HCV genotype 1-infected patients who had been treated with combination therapy after initial failure to respond permanently to interferon alone. Our results show that during the early treatment phase mutation rate is not enhanced during combination therapy and that, at least in the major variant, shifts in the NS5A domain resulting in the occurrence of viral variants, which are more interferon-sensitive, do not occur.