To document the transmission of hepatitis C virus (HCV) through needlestick accidents, 3 health workers who acquired HCV through such accidents and their HCV donor patients were studied using molecular evolutionary analysis based on the HCV E2 region. At least six clones were sequenced from each subject. Nucleotide substitutions were estimated by the six-parameter method, and a phylogenetic tree was constructed by the neighbor-joining method. HCV isolates from the donor patient and the recipient were nested in one monophyletic cluster; this clustering was confirmed to be statistically significant by bootstrap analysis. The nucleotide divergence among the isolates from the recipient was always smaller than that from the donor, supporting the notion that the direction of transmission was from the donor to the recipient. These findings provide evidence, at a molecular evolutionary level, that HCV was transmitted through needlestick accidents.