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(TM) 2.0 and RIBA(TM) 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(TM) 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(TM) 3.0 negative (PCR positive), 1 was indeterminate (c33c, PCR positive), and 33 were positive (27 PCR positive) by RIBA(TM) 3.0. Thus only 1 PCR-positive patient was negative with RIBA(TM) 2.0 and 3.0 assays. Two of the 3 RIBA(TM) 2.0 indeterminate samples were positive with RIBA(TM) 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(TM) 3.0 test may improve the sensitivity for the detection of antiHCV. Nevertheless, in potentially immunocompromised patients undergoing dialysis, PCR continues to be the only reliable test for detecting viremia.