The newly developed anti-HCV assays AxSYM HCV version 3.0 and IMx HCV version 3.0 were evaluated with regard to their precision, sensitivity and specificity in comparison to the HCV EIA 3.0 (Abbott GmbH, Wiesbaden, Germany). Precision testing was undertaken using five positive controls with different anti-HCV levels for each assay. Specificity was estimated by testing 4383 blood donor specimens. The supplemental assay Matrix HCV 2.0 (Abbott GmbH, Wiesbaden, Germany) was used to confirm repeatedly reactive results. Samples which had been found to be positive or indeterminate by Matrix HCV 2.0 were tested by qualitative polymerase chain reaction after reverse transcription (RT-PCR, Amplicor HCV test, Roche Diagnostic Systems, Basel, Switzerland). To determine sensitivity, 20 commercially available seroconversion panels were tested. Based on supplemental testing, the apparent specificities of AxSYM HCV version 3.0, IMx HCV version 3.0 and HCV EIA 3.0 were estimated to be 99.84, 99.98 and 99.80%, respectively. In seroconversion panel testing, AxSYM and IMx HCV version 3.0 detected seroconversion in up to 12/20 panels earlier and in up to 1/20 cases later than the comparison EIA. The highest sensitivity was shown in AxSYM HCV version 3.0, followed by IMx HCV version 3.0 and HCV EIA 3.0. Based on the improved seroconversion sensitivity and specificity, the AxSYM and IMx HCV version 3.0 assays appear to be suitable for detecting HCV antibodies in blood donor testing and other routine laboratory assessments.