The genomic RNA of Hepatitis C virus (HCV) contains a single large open reading frame for virion structural proteins and viral nonstructural polypeptides. HCV NS3 protein (p70) was predicted to be a serine-type proteinase, a nucleotide triphosphatase (NTPase), and an RNA helicase based on the presence of sequence motifs. We demonstrated that the NS3 protein expressed in insect cells possesses an NTPase activity. Employing the baculovirus system, we partially purified the NS3 protein fused to 6-histidine residues using metal-binding chromatography. It is revealed that an analog of p70, p47 was also expressed. The p47 protein did not seem to contain an N-terminal serine protease domain of the p70 protein, because p47 did not bind to a metal-binding column. The elutes containing p70 showed a specific NTPase activity. This activity was not found in the other elute fractions. NTPase activity was completely inhibited by the murine NS3 specific antibodies. Above results strongly suggest that NS3 protein of HCV contains NTPase activity.