Hepatitis C virus (HCV) infection is endemic in long-term dialysis units. We assessed the performance of a recently developed HCV 3.0 assay for the detection of HCV antibodies in patients undergoing dialysis. The study evaluated 128 patients undergoing long-term maintenance hemodialysis. Anti-HCV was detected by 2.0 and 3.0 enzyme immunoassay (EIA). Results were confirmed with recombinant immunoblot assays (RIBA 2.0 and RIBA 3.0). HCV RNA was detected by using reverse transcriptase-polymerase chain reaction (RT-PCR). Thirty-two patients (25%) were HCV EIA 2.0 positive. Of these, 1 was RIBA 2.0 negative (PCR positive), 3 were indeterminate (3 PCR positive), and 28 were positive (23 PCR positive). Thirty-five (27%) were HCV EIA 3.0 positive. One was RIBA 3.0 negative (PCR positive), 1 was indeterminate (c33c, PCR positive), and 33 were positive (27 PCR positive) by RIBA 3.0. Thus only 1 PCR-positive patient was negative with RIBA 2.0 and 3.0 assays. Two of the 3 RIBA 2.0 indeterminate samples were positive with RIBA 3.0. One remained indeterminate but was HCV RNA positive. In summary, HCV 3.0 EIA detected 4 additional viremic patients but was positive in 6 PCR-negative subjects. A high correlation of the presence of antibody to c33c with HCV RNA (28 of 34, 82%) was found, and it was found in all anti-HCV positive samples and in 1 indeterminate sample. We conclude that the HCV EIA 3.0 test with the supplemental confirmatory RIBA 3.0 test may improve the sensitivity for the detection of anti-HCV. Nevertheless, in potentially immunocompromised patients undergoing dialysis, PCR continues to be the only reliable test for detecting viremia.