GB virus C/hepatitis G virus (GBV-C/HGV), a recently discovered orphan flavivirus, is distantly related to hepatitis C virus (HCV). Although both GBV-C/HGV and HCV can be transmitted by the parenteral route, their principal modes of transmission and associated risk behaviors may differ. Using reverse transcription-polymerase chain reaction, the 5'-noncoding regions of GBV-C/HGV and HCV were amplified from plasma or sera of 209 individuals infected with human immunodeficiency virus type 1 (HIV-1). As verified by Southern blot analysis, GBV-C/HGV and HCV infection were detected in 37 (17.7%) and 22 (10.5%) of 209 HIV-1-infected individuals, respectively. GBV-C/HGV infection was significantly associated with homosexual sex (P = 0.044) and was more common than HCV infection among HIV-1-infected homosexual men (P = 0.006). The prevalence of GBV-C/HGV infection was nearly equal in women infected with HIV-1 via high-risk heterosexual sex (14.0%) or injection drug use (IDU) (17.5%). By contrast, HCV infection was associated significantly with women reporting IDU when compared to women reporting high-risk heterosexual sex (P < 0.0001). Alanine aminotransferase levels were elevated in HIV-1-infected individuals who were co-infected with HCV (P = 0.009), but not with GBV-C/HGV (P = 0.9). The high prevalence of GBV-C/HGV infection in HIV-1-infected nondrug-injecting homosexual men and among women engaging in high-risk heterosexual sex is consistent with transmission by the mucosal route and with acquisition of infection by the receptive rather than insertive partner.