The latest prevalence of hepatitis C virus (HCV) infection was evaluated in 548 Japanese patients undergoing hemodialysis, and 216 members of the hospital dialysis staff. An assay of anti-HCV antibodies was performed using both first- and second-generation immunoenzymatic tests, and anti-HCV specificity was confirmed with a second-generation recombinant immunoblot assay. Of 548 hemodialysis patients, 90 (16.4%) were positive with first-generation, and 166 (30.3%) with second-generation tests. These rates were significantly higher than those for either hospital staff members (0.5% and 2.3%; p < 0.01) or healthy blood donors (0.98% and 1.30%; p < 0.001). Patients with a history of blood transfusion tended to have a higher positivity rate for anti-HCV than did the non-transfused group (35.4% vs. 25.7% with second generation tests; p < 0.05). Positivity for anti-HCV was related to the duration of hemodialysis. Only a few patients seroconverted during the 2-year, 5-month period between the previous and present studies. Although hemodialysis patients remain a high-risk group for HCV infection, the prevalence of anti-HCV antibodies has decreased recently thanks to the use of erythropoietin for renal anemia, the universal screening of blood donors for anti-HCV antibodies, and improvements in infection control measures for this virus.