In order to determine the significance of anti-hepatitis C virus (anti-HCV) antibodies in blood donors, 46 consecutive asymptomatic individuals were recruited at the blood bank of Hospital São Paulo, São Paulo, Brazil. They were submitted to an interview to collect epidemiological data and to clinical examination and blood samples were obtained for biochemical, serological and virological analysis. All patients were followed for a minimum period of six months and those with abnormal mean alanine aminotransferase (ALT) levels were submitted to a liver biopsy after giving informed consent. Hepatitis C virus RNA (HCVRNA) was detected by the polymerase chain reaction (PCR) in 22/47 (47.8%) patients and this finding was associated with parenteral risk factors (P = 0.03) and ethanol abuse (P = 0.03). HCVRNA positivity was also associated with abnormal levels of ALT (P < 0.001) and gamma-glutamyl transpeptidase (gamma-GT) (P = 0.01). Abnormal ALT levels were good marker of viremia, with 86.4% sensitivity and 79.2% specificity. Twenty-three patients with elevated mean ALT levels were submitted to a liver biopsy and histopathological changes were observed in 17 of them (73.9%). HCVRNA positivity was associated with severe forms of hepatic disease (chronic hepatitis and cirrhosis). These results indicate the need for a judicious evaluation of all anti-HCV-positive blood donors, including clinical examination, biochemical tests and liver histology when ALT is persistently elevated.