BACKGROUND : Current diagnosis of hepatitis C virus (HCV) infection requires two sequential steps: testing for anti-HCV followed by HCV RNA PCR to confirm viremia. We have developed a highly sensitive and specific HCV-antigens enzyme immunoassay (HCV-Ags EIA) for one-step diagnosis of viremic HCV infection. OBJECTIVE : To assess the clinical application of the HCV-Ags EIA in one-step diagnosis of viremic HCV infection in human immunodeficiency virus (HIV)-coinfected individuals. METHODS : The study blindly tested HCV-Ags EIA for its performance in one-step diagnosing viremic HCV infection in 147 sera: 10 without HCV or HIV infection; 54 with viremic HCV monoinfection; 38 with viremic HCV/HIV coinfection; and 45 with viremic HCV and non-viremic HIV coinfection. RESULTS : Upon decoding, it was 100% accordance of HCV-Ags EIA to HCV infection status by HCV RNA PCR test. In five sera with HCV infection, HCV RNA was as low as 50-59 IU/mL, and four out of five tested positive for HCV-Ags EIA. Likewise, it was also 100% accordance of HCV-Ags EIA to HCV infection status by HCV RNA PCR in 83 sera with HCV and HIV coinfection, regardless if HIV infection was active or not. CONCLUSIONS : The modified HCV-Ags EIA has a lower detection limit equivalent to serum HCV RNA levels of approximately 100 IU/mL. It is highly sensitive and specific in the setting of HIV coinfection, regardless of HIV infection status and CD4 count. These data support the clinical application of the HCV-Ags EIA in one-step diagnosis of HCV infection in HIV-infected individuals.