We tested serum samples from 99 patients undergoing maintenance hemodialysis for hepatitis C virus (HCV) antibodies using a first-generation, licensed anti-HCV enzyme immunoassay (EIA) and a second-generation anti-HCV EIA that detect three gene products c100-3, NS3, and core. Specimens that were repeatedly reactive by either or both screening assays were further evaluated by testing with supplemental EIAs and a dot blot immunoassay. There was 87.9% agreement between the licensed HCV EIA and the HCV EIA second generation. HCV EIA Second Generation detected 10 more positive specimens than HCV EIA, for an increase in detection from 33.3% (33/99) to 43.4% (43/99). We conclude that HCV EIA Second Generation improves detection of HCV infection in hemodialysis patients.