Abstract Background Early diagnosis of hepatitis C virus (HCV) infection in HIV-infected patients has significant implications for patient management. However, the currently recommended serological screening strategy for identifying such patients could be improved. Little is known about the performance of routine antibody-only tests, compared with newer serological antigen–antibody detection assays and nucleic acid testing in this patient group. Objectives To compare the performance of antibody and antigen–antibody detecting methods with nucleic acid testing in the diagnosis of acute HCV in HIV-infected individuals. Study design 123 samples from 25 HIV-infected patients with acute HCV infection were tested retrospectively. The time of infection was estimated. The performance of antibody, antigen–antibody and nucleic acid detecting methods in diagnosing acute HCV infection was assessed and the sensitivity of the assays compared. Results Only 20% of samples that were positive for HCV RNA were simultaneously positive for HCV antibody. In contrast, 68% of the total number of samples were positive and 32% negative by the antigen–antibody assay. Patients became antibody-positive on average 7 months after HCV RNA was detected. Conclusion In HIV-infected patients nucleic acid testing is the most sensitive means of diagnosing acute HCV C infection. A serological assay offering combined detection of antibody and antigen enhances sensitivity of detection, compared to antibody-only assays.