The hepatitis C virus (HCV) NS3 protein possesses three enzymatic activities: an N-terminal serine protease activity, a C-terminal RNA-stimulated NTPase activity, and an RNA helicase activity. To characterize them, the full-length NS3(631)/4A and three C-terminal truncated proteases (NS3(201)/4A, NS3(181)/4A, and NS3(155)/4A were expressed in mammalian cells with HSV amplicon-defective viruses. Our results revealed that all of the NS3/4A proteins produced in mammalian cells (except NS3(155)/4A) are active in processing both cis and trans cleavage sites. Temperature optimization studies revealed that the protease is more active at temperatures ranging from 4 to 25 degrees C and is completely inactive at 42 degrees C. The RNA-stimulated ATPase activity was characterized with a partially purified NS3(631)/4A fraction and has a higher optimal temperature at 37 to 42 degrees C. The effects of detergents on both NS3 protease and RNA-stimulated ATPase were similar. Nonionic detergents such as Triton X-100, Nonidet P-40 and Tween 20 did not affect the activities, while anionic detergents such as sodium dodecyl sulfate and deoxycholic acid were inhibitory. Zwitterionic detergent such as 3-[(3-cholamidopropyl)- dimethyl-ammoniol-1-propanesulfonate (CHAPS) inhibited protease activity at a concentration of 0.5% (8 mM), which had no effect on ATPase activity. Finally, RNA-unwinding activity was demonstrated in the NS3(631)/4A fraction but not in the similarly purified NS3(181)/4A and NS3(201)/4A fractions. NS(363)/4A unwinds RNA duplexes with 3' but not 5' single-stranded overhangs, suggesting that the NS3 RNA helicase functions in a 3'-to-5' direction.