Background: Testing for hepatitis C virus core antigen (HCV Ag) may represent a complementary tool to anti-HCV and HCV-RNA in the diagnosis and monitoring of HCV infection. Objective: To evaluate the performance characteristics of the automated Abbott ARCHITECT HCV Ag assay. Study design: Five sites analyzed over 3000 routine serum samples from populations at different risk, comparing HCV Ag results with anti-HCV screening and supplemental assay results and with HCV-RNA. Results: The HCV Ag assay showed a specificity of 100%, a good precision (CV < 10%) and excellent dilution linearity (r > 0.999). The sensitivity (3 fmol/L) corresponds to 700–1100 IU/mL of HCV-RNA. A non-linear correlation with HCV-RNA was found: r = 0.713 vs. Siemens bDNA (523 specimens), r = 0.736 vs. Roche Cobas TaqMan (356 specimens) and r = 0.870 vs. Abbott Real-Time PCR (273 specimens). HCV Ag quantitation was equally effective on different HCV genoypes (239 for genotype 1/1a/1b/1c, 108 for genotype 2/2a/2c, 86 for genotype 3/3a, 50 for genotype 4/4a/4c/4d). Testing of subjects at high risk for HCV and with potential or actual impairment of the immune system identified 2 cases negative for anti-HCV and positive for HCV Ag on 361 hemodialyzed (0.6%) and 7 cases on 97 (7.2%) among transplant recipients. HCV Ag positivity anticipated anti-HCV seroconversion in all three cases of acute hepatitis C. Conclusions: HCV Ag may be used as reflex testing on anti-HCV positive individuals to confirm or exclude an active infection, and on subjects with acute hepatitis or belonging to high risk groups.