Hepatitis C virus (HCV) infection is the cause of significant long-term morbidity and mortality. While often asymptomatic, the majority of HCV infections result in chronic hepatitis that can progress to cirrhosis, end-stage liver disease, and hepatocellular carcinoma. It is estimated that about 170 million people worldwide, approximately 3% of the world's population, are infected with HCV (45). Presently, there is no specific antiviral agent directed against HCV and no vaccine for prevention of hepatitis C infection. The current approved treatments, interferon monotherapy or interferon in combination with ribavirin, have limited benefits. Interferon alone achieves a sustained viral response in only 10 to 20% of patients (32), while the recommended therapy of pegylated interferon in combination with ribavirin results in a sustained viral response in 54% of patients (23). The response rate is lower in patients who are infected with HCV genotype 1b (ϳ34%) (23,47). Adverse side effects, such as severe flu-like symptoms, depression, psychoses, and anemia, are associated with these treatments, causing approximately 20% of patients to discontinue therapy (13,17,33). Consequently, there is an urgent need for the development of an HCV-specific antiviral that is more effective, less toxic, and easier to administer than the present therapy.HCV, a member of the Flaviviridae family, is a positivesense, single-stranded RNA virus with a genome size of ϳ9.4 kb (26, 39). The genome RNA encodes a polyprotein of 3,010 to 3,011 amino acid residues in the order NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH. This polyprotein is processed by host and viral proteases (18,34). The nonstructural protein 5B (NS5B) is a virus-encoded RNA-dependent RNA polymerase (RdRp) that is responsible for replication of the viral RNA genome. A functional counterpart of NS5B does not exist in mammalian cells. For this reason, an inhibitor of NS5B could serve as an effective and selective agent for treating HCV infection.The enzymatic activity of the NS5B enzyme in vitro has been extensively characterized (9,19,21,40). Both the full-length and carboxyl-terminally truncated forms of NS5B have been shown to be functionally active in the presence of HCV or exogenous RNA templates. Although HCV replicates inefficiently in cell culture, viral replication, including the activity of the NS5B polymerase, can be studied in a cell culture system