The NS3 protein of hepatitis C virus (HCV) is thought to be essential for viral replication. The N-terminal domain of the protein contains protease activity and the C-terminal domain contains nucleotide triphosphatase and RNA helicase activity. The RNA helicase domain of HCV NS3 protein was purified by using affinity-column chromatographic methods, and crystallized by using the microbatch crystallization method under oil at 277 K. The crystals belong to primitive trigonal space group P3(1)21 or P3(2)21 with cell dimensions of a = b = 93.3, c = 104.6 Angstrom. The asymmetric unit contains one molecule of the helicase domain, with the crystal volume per protein mass (V-m) of 2.50 Angstrom(3) Da(-1) and solvent content of about 50.8% by volume. A native data set to 2.3 Angstrom resolution was obtained from a frozen crystal indicating that the crystals are quite suitable for structure determination by multiple isomorphous replacement.