Recent studies have shown that rapid, instrument-free assays for the detection of antibody to human immunodeficiency virus (HIV) can be as sensitive and specific as enzyme-linked immunosorbent assay (ELISA) for screening of donated blood in developing countries. Currently, however, specimens which test positive on a screening assay must still be confirmed by Western blot (immunoblot), a method which is not feasible in most developing-country laboratories. We examined whether a testing hierarchy which utilizes neither conventional ELISA nor Western blot can be reliably used for screening and confirmation of HIV infection in a high-risk population. In a retrospective analysis of 3,878 specimens which were screened for antibody to HIV in Kinshasa, Zaire, we observed that a testing hierarchy consisting of duplicate HIVCHEK screening assays followed by duplicate Serodia-HIV confirmatory assays resulted in correct confirmation of all ELISA- and Western blot-positive specimens. We conclude that such a testing hierarchy can produce highly accurate results for identification of positive specimens in routine HIV testing and provides a practical alternative to conventional methods of HIV screening and confirmation.