The Hepatitis C Virus (HCV) NS3 protein contains amino acid motifs of a serine proteinase, a nucleotide triphosphatase (NTPase), and an RNA helicase based on amino acid sequence analysis. Proteinase and NTPase activities of the HCV NS3 protein were reported by several investigators. Here, we show that the recombinant HCV NS3 protein purified from a T7 promoter and His-tag expression system possesses an RNA helicase activity. The recombinant HCV NS3 protein consists of 466 amino acids from the carboxy terminal of a HCV NS3 open reading frame and 25 additional residues from the vector. The recombinant HCV NS3 protein was purified by metal-binding chromatography. The helicase activity requires ATP and divalent cations such as Mg2+ and Mn2+. The helicase activity was abolished by monoclonal antibody specific to the HCV NS3 protein.