The objective of this study was to validate the use of OraQuick ADVANCE Rapid HIV-1/2 Antibody test (OraSure Technologies Inc., Bethlehem, PA) on oral fluid for a population-based HIV prevalence survey of rural youth in southeast Zimbabwe. The evaluation was conducted in patients presenting for voluntary counseling and testing at rural clinics. Each participant provided an oral fluid sample tested using OraQuick ADVANCE. In addition, dried blood specimens were collected and tested blind at the National Microbiology Reference Laboratory in Harare using two enzyme-linked immunosorbent assays (ELISA; Vironostika, Biomérieux BV, Boxtel, The Netherlands and Ani Labsystems, Ltd., Vantaa, Finland) with confirmatory Western blot (MP Diagnostics [formerly Genelabs Diagnostics], Medical Technology Promedt Consulting GMBH, St. Ingbert, Germany) for samples with discrepant results. Diagnostic accuracy of the oral fluid assay was determined against the ELISA/Western blot algorithm as gold standard. Five hundred and ninety-one participants took part in the study between February and July 2006. Sensitivity of the test on oral fluid was 100% (95% confidence interval [CI]: 97.9-100), and specificity was 100% (95% CI: 99.1-100). HIV prevalence based on the reference standard was 29.8% (95% CI: 26.1-33.5). This is one of the first validations of this rapid assay on oral fluid conducted in a general population to be reported in Africa. While there are some limitations with the assay (e.g., unlikely to detect those in early stages of HIV infection or with reduced viral load; altered accuracy in pregnancy) these limitations also apply to other rapid assays. The results showed the assay to be 100% accurate in determining HIV status, performed well in field settings, and can be considered suitable for use in epidemiologic surveys aiming to estimate HIV prevalence in general populations.