In November 1989, the Japanese Red Cross began screening blood donors for the hepatitis C virus antibody (anti-HCV) by first-generation assay and high-titer hepatitis B virus core antigen antibody. A significant reduction in the incidence of acute posttransfusion hepatitis was reported; however, the incidence still ranged from 2% to 4%. The Red Cross changed to the second-generation assay in February 1992, the objective being the complete elimination of potential posttransfusion hepatitis. The aim was to elucidate the advantage of second-generation assay as a blood-donor screening test. The incidence of posttransfusion hepatitis after the introduction of second-generation assay was compared with that before the introduction of the first-generation assay and with that during its use. The incidence of posttransfusion hepatitis was 9.6% (216/2,240) before anti-HCV-s donor screening. It was 3.7% (24/655) and 0.9% (3/326) after the introductions of the first-and second-generation hepatitis C virus (HCV) assays, respectively (χ2 = 50.0, P < .01). Blood-donor screening by second-generation anti-HCV provided a significant benefit compared with the first-generation assay.