The performance of a fully automated, random access, enhanced chemiluminescence immunoassay (Ortho/ ECi) for the detection of antibody to hepatitis C virus (HCV) (anti-HCV), HBsAg, and antibody to HBsAg (anti-HBsAg), in human serum was compared to a Abbott second-generation enzyme immunoassay (EIA 2.0). The Ortho/ECi assays employ an immunometric technique with enhanced chemiluminescence for optimal assay performance. With regard to the study of clinical laboratory performance, six groups of sera prescreened with Abbott EIAs were assayed: anti-HCV-negative samples (n ؍ 318), anti-HCV-positive samples (n ؍ 177), anti-HBsAg-negative samples (n ؍ 241), anti-HBsAg-positive samples (n ؍ 239), HBsAg-positive samples (n ؍ 158), and HBsAg-negative samples (n ؍ 312). Sera with discrepant results in the two serological assays were resolved by confirmatory tests. Sera with indeterminate results by one or more confirmatory tests were evaluated by reviewing medical records. The overall concordance between the Ortho/ECi assay and the Abbott EIA were 97.78, 93.54, and 97.66% for anti-HCV antibodies, anti-HBsAg antibodies, and HBsAg, respectively. After resolving the discrepancies, the specificities of the new assay for anti-HCV and anti-HBsAg antibodies and HBsAg were 98.1, 92.8, and 100%, respectively. The sensitivities of the new assay for anti-HCV, anti-HBsAg, and HBsAg were 100, 98.8, and 97.4%, respectively. In conclusion, The Ortho/ECi assays for diagnosis of HCV and hepatitis B virus (HBV) infections are highly specific and sensitive assays. The rapid turnaround time, random access, full automation, and high throughput make it an effective assay system for clinical laboratory diagnosis of HCV and HBV infections.The four immunoassays described here are associated with the diagnosis of hepatitis C and B virus (HCV and HBV, respectively) infections. HCV, an enveloped positive-stranded RNA virus of the Flaviviridae family, has been demonstrated to be the etiologic agent of 90% of chronic non-A, non-B hepatitis (2, 5, 10, 15). HCV infection is often asymptomatic; however, the vast majority of HCV-infected individuals (Ͼ85%) develop persistent chronic infection and chronic hepatitis (9,32,45,46). Diagnosis of HCV infection has serious implications, especially for the high-risk patients such as those undergoing hemodialysis (20,27,35). The presence of anti-HCV antibodies indicates that an individual may have been infected with HCV and/or may be capable of transmitting HCV infection while active infection is marked by the presence of HCV RNA detected by reverse transcriptase PCR (6,17,35,42). Detection of viremia is often required in certain cases of acute infection and/or immunodeficiency, where individuals may fail to produce antibodies specific for HCV (32, 39).The HCV genome consists of seven functional regions: the core, the envelope, including the E1 and E2 regions, and the nonstructural region, including NS2, NS3, NS4, and NS5 (31). The first commercially available HCV test was an enzymelinked immunosorbent a...