The prevalence of hepatitis C virus (HCV) antibodies in 2,557 asymptomatic volunteer Brazilian blood donors is reported. Using the line immunoassay (Inno-LIA) as a confirmatory test on ELISA anti-HCV-positive reacting sera, a prevalence rate of 2.7% for anti-HCV positivity was found. By comparison, prevalences of 1.6% for hepatitis B surface antigen, 0.9% for Treponema pallidum, 0.4% for human immunodeficiency virus and 0,04% for Trypanosoma cruzi were observed. Only 57% of the HCV-positive donors had elevated alanine aminotransferase (ALT) levels. Using previous criteria, based on surrogate markers (ALT ≥ 50 IU/1 and for anti-hepatitis B core antibody), for HCV infection at that time, only 25% of the HCV-positive donations would have been eliminated. In view of the high prevalence of anti-HCV reactivity among the Brazilian blood donor population and the poor reliability of surrogate markers, it is recommended that routine screening for anti-HCV in Brazilian blood donors is introduced.