Markers of viral replication are fundamental tools for understanding the mode of transmission, diagnosis and management of hepatitis C virus (HCV) infection. A new enzyme-linked immunosorbent assay for quantitative detection of free and complexed HCV core antigen (HCV Ag) has been developed. The aim of this study is to evaluate the clinical performance of the new test and compare it with the most widely used commercially available RT-PCR-based assay. To determine the cut-off value we tested 60 samples from anti-HCV negative samples and selected a qualitative cut-off value of 3 pg/ml. To evaluate the usefulness of the new assay in confirming serologically indeterminate results we collected 62 sera. To evaluate the HCV Ag and HCV-RNA relationship we tested 245 samples from patients with different clinical conditions. The results of 61 out of 62 (98.4%) anti-HCV indeterminate samples were found to agree, whereas only one serum was found to be RT-PCR positive and HCV Ag negative. We also found the results to agree in 77.6% (190/245) of the samples from infected patients, while we observed higher agreement in untreated patients, both with and without evidence of liver damage. The correlation coefficient (r) observed between HCV Ag and HCV-RNA was 0.88. The regression line meets the cut-off value at an HCV-RNA concentration of approximately 40,000 IU/ml. In conclusion, we found that the results from the total HCV Ag test agree with the RT-PCR results and therefore we believe that this test could become a useful tool for the diagnosis and management of HCV infection.