The biochemical properties of the RNA-dependent RNA polymerase (RdRp) of the hepatitis C virus were analyzed. A hexahistidine affinity-tagged NS5B fusion protein was expressed with recombinant baculoviruses in insect cells and purified to near homogeneity. Enzymatic activity of the purified protein was inhibited by KCl or high concentrations of NaCl and was absolutely dependent on Mg2+, which could be replaced by Mn2+. NS5B was found to be processive and able to copy long heteropolymeric templates with an elongation rate of 150-200 nucleotides/min at 22 degreesC. Kinetic constants were determined for all four nucleoside triphosphates and different templates. In case of a heteropolymeric RNA template corresponding to the last 319 nucleotides of the hepatitis C virus genome, Km values for UTP, GTP, ATP, and CTP were approximately 1.0, approximately 0.5, approximately 10, and approximately 0.3 microM, respectively. The profile of several inhibitors of RdRp activity and substrate analogs indicated that the enzyme has a strong preference for ribonucleoside 5'-triphosphates and that it closely resembles 3Dpol of picornaviruses.