Background. Chronic seronegative hepatitis C virus (HCV) infection is defined as being HCV antibody (anti-HCV) negative, but HCV RNA positivity occurs in individuals infected with human immunodeficiency virus (HIV). However, associated factors are not well established because of the small number of reported cases. Methods. Multivariate logistic regression analysis of HIV-infected subjects from 4 cohorts (Tien et al., 2006; Bonacini et al., 2001; George et al., 2002; and Hall et al., 2004) determined factors associated with HCV RNA positivity in anti-HCV-negative subjects. HCV enzyme immunoassay 2.0 was used to determine anti-HCV status. Results. Among 1174 anti-HCV-negative, HIV-infected subjects, the prevalence of seronegative HCV infection was 3.2% (95% confidence interval [CI], 2.2%-4.3%). History of injection drug use (IDU; OR, 5.8; 95% CI, 2.7-12.8), higher alanine aminotransferase (ALT) level (OR, 2.0 per doubling; 95% CI, 1.3-3.2), and CD4 cell count <200 cells/μL (OR, 2.3; 95% CI, 1.1-4.8) were associated with HCV RNA positivity in anti-HCV-negative subjects. Among those with a history of IDU who had either a CD4 cell count <200 cells/μL or an ALT level greater than the upper limit of normal, the prevalence of seronegative HCV infection was 24% (95% CI, 13%-39%). Conclusions. Detectable HCV RNA in the context of a negative HCV enzyme immunoassay 2.0 result in HIV-infected patients is low, but higher than the reported prevalence in HIV-uninfected patients. Our findings suggest that HCV RNA testing should be performed in anti-HCV-negative, HIV-infected patients, especially those with a history of IDU and either a CD4 cell count <200 cells/μL or an abnormal ALT level. © 2007 by the Infectious Diseases Society of America. All rights reserved.