We previously reported the relationship between viral polymerase polymorphisms and the initial decline in viral load induced by interferon-alpha and ribavirin therapy in genotype 1b-related chronic hepatitis C patients. The presence of E124K and I85V of NS5B was closely associated with viral clearance at 8 weeks of treatment. The aim of this study was to investigate the mechanisms by which this polymorphism of NS5B protein affects early viral clearance. We used a replicon system derived from strain O, genotype 1b virus. Three mutants (V85I), (K124E), and (V85I/K124E) were introduced to the replicon. OR6c, a derivative of HuH7 cells, was transfected with the replicon including a luciferase reporter gene. Luciferase activities were measured 72 hr post-transfection. All three mutants showed higher luciferase activity than that of the wild type, and the V85I mutant showed the highest activity. This result was also confirmed by neomycin gene-containing replicons with same mutations. All replicons were down-regulated by ribavirin, but the level of reduction in the V85I mutant was the lowest. Our results suggested that this mutation at least partly contributes to resistance to early viral clearance during interferon and ribavirin combination therapy.