We studied 102 consecutive subjects after their completion of acute lymphoblastic leukemia (ALL)-directed chemotherapy, for evidence of hepatitis C virus (HCV) infection by enzyme immunoassay 2 and 3, second generation recombinant immunoblot assay and reverse transcription-polymerase chain reaction (PCR) for detection of circulating HCV-RNA. Forty-four patients (43%) had evidence of exposure to HCV; 30 of these were anti-HCV+. Of the 23 patients who were positive for both anti-HCV and HCV-RNA, 16 (69%) had a moderate increase in serum alanine aminotransferase (ALT) activity without clinical signs of liver disease. Fourteen patients were seronegative despite the presence of HCV-RNA in the serum. The proportion of different HCV genotypes was not significantly different from other anti-HCV+ patient groups. Although half of the patients with genotype III had normal ALT value, patients with normal ALT levels were represented in all genotype groups. Our study documents the prevalence of HCV infection in childhood ALL survivors, which is responsible for the majority of cases of non-B chronic liver disease in these patients. Whereas serologic screening identifies over 70% of patients with ongoing HCV infection, real HCV infection may be present even in the absence of a detectable humoral immune response to the virus. Based on this observation, determination of HCV-RNA by PCR should be recommended in patients in prolonged remission even if they test negative on serological assay. Normal ALT levels do not exclude the presence of HCV infection because the values were repeatedly normal in over half of our viremic patients.