Co-infection with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) is a significant global health problem. The two viruses are transmitted with high efficacy via blood-to-blood contact, mainly intravenous drug use (IVDU), whereas HCV is less easily transmitted sexually. Antibody testing is the main screening method for HCV infection, although it may not be the optimal option for HIV infection. The aim of this study was to investigate HCV infection in HIV-positive patients, with and without a detectable anti-HCV antibody response. A total of 187 plasma samples were obtained from HIV-positive patients in Surabaya, Indonesia and examined for anti-HCV [HCV enzyme immunoassay (EIA) 3.0], HCV genotype/subtype [reverse transcription-polymerase chain reaction (RT-PCR) using primers targeting a part of NS5B/5'UTR followed by sequencing] and HCV viral load (quantitative RT-PCR). A total of 119 patients (63.6%) were found to be anti-HCV-positive and, among these, HCV RNA was detected in 73 (61.3%), with HCV-1a as the predominant subtype (31.5%). Of the 68 anti-HCV-negative samples, HCV RNA was detected in 26/68 (38.2%) mostly as the HCV-3a subtype (50%). High HCV viral loads were more common among the HCV-seropositive patients. The HCV-seropositive samples with detected HCV RNA were mostly obtained from HIV-positive patients with parenteral transmission (IVDU) (76.7%); however, the HCV-seronegative samples with detected HCV RNA were mostly from patients who had acquired HCV through heterosexual transmission (61.5%). In conclusion, HIV-positive patients were at high risk of becoming co-infected with HCV and several remained HCV-seronegative. Furthermore, there may exist differences in HCV seropositivity and subtypes between HIV-positive patients who acquired HCV sexually and those who acquired HCV parenterally.