The nonstructural 5B (NS5B) protein of hepatitis C virus (HCV) is an RNA-dependent RNA polymerase (RdRp) with a key role in HCV replication. To characterize the functional roles of NS5B in HCV replication, we produced a panel of 10 monoclonal antibodies (mAbs) directed against NS5B protein from mice immunized with functionally active RdRp. The epitopes of eight mAbs are localized in the middle region (amino acid 240-263) of NS5B protein. On the other hand, the epitopes of two mAbs are mapped to amino acids 67-88 at the N-terminus of NS5B protein. To examine the effects of mAbs on HCV-RNA replication, we performed in vitro RdRp assay using either the 3'-untranslated region (UTR) or the full-length of HCV-RNA as a template in the presence of each mAb. mAbs specific for the middle region of NS5B had no effect on RdRp activity. Surprisingly, mAb recognizing the N-terminal region of NS5B inhibited RdRp activity in a dose-dependent manner. We have confirmed the same result using the other subclass of mAb, whose epitope is also localized to the same N-terminal region of NS5B. These data show that NS5B contains a B-cell epitope located between amino acid residues 67 and 88. Binding of this epitope with an antibody interferes with the enzymatic function of NS5B.