The EIAgen HCV test (Adaltis Inc., Montreal, Canada) is an enzyme immunoassay (EIA) for the detection of anti-hepatitis C virus (HCV) antibodies. This study compared the performance of this test side-by-side with the current Ortho HCV 3.0 Anti-HCV assay (Ortho-Clinical Diagnostics Inc., Johnson & Johnson Company, Raritan, NY, USA). Among 2559 specimens examined, 178 were true positives, 2376 were true negatives and 5 were indeterminate. The sensitivity of the EIAgen HCV test was 100%, versus 98.3% for the Ortho HCV test, while their respective specificities were 98.1% and 98.2%. The EIAgen HCV test gave a positive predictive value of 79.8% and a negative predictive value of 100%. Overall, the concordance of this test with the Ortho HCV test was 98.2%. Specimens from potentially interfering substances, such as sera from pregnant women, sera from patients with acute non-C hepatitis, autoimmune diseases, lipidemia, or from patients undergoing hemolysis, showed no interference with either EIA. An EIAgen HCV test signal-to-cut-off ratio of >5.9 would be highly predictive of a true-positive finding in these specimens. The EIAgen HCV test is well suited for screening blood and blood products in antibodies to HCV.