We longitudinally studied 51 patients from two hemodialysis centers to determine the prevalence of hepatitis C virus infection in hemodialysis patients. Serum samples were tested for antibody to HCV by first- and second-generation enzyme immunoassays and for hepatitis C virus RNA by nested polymerase chain reaction assay. Antibody to hepatitis C virus was detected in the initial serum samples by first-generation enzyme immunoassay and second-generation enzyme immunoassay in 6 (11.8%) and 11 (21.6%) patients, respectively. First-generation enzyme immunoassay had a false-positive rate of 33.3% and a false-negative rate of 63.6%. Hepatitis C virus RNA was found in eight second-generation enzyme immunoassay-positive patients (72.7%) and in one patient negative for antibody to hepatitis C virus (2.5%) giving an overall positivity rate of 17.6%. After 19 mo, antibody to hepatitis C virus was detected in 15 patients (29.4%) on second-generation enzyme immunoassay; hepatitis C virus RNA was found in 13 patients (25.5%). Hepatitis C virus markers persisted in all 12 patients with initial evidence of hepatitis C virus infection. Three patients acquired hepatitis C virus infection during the interim, giving a new infection rate of 4.9% per patient-year. Antibody to hepatitis C virus, hepatitis C virus RNA or both was detected in 55.6% of patients with biochemical changes suggestive of non-A, non-B hepatitis. Of the 15 antibody to hepatitis C virus, second-generation enzyme immunoassay-positive patients, 66.7% had persistently normal serum transaminase levels. In summary, hepatitis C virus infection is common among hemodialysis patients. First-generation enzyme immunoassay is an unreliable assay for antibody to hepatitis C virus in these patients.(ABSTRACT TRUNCATED AT 250 WORDS)