Effective health care delivery to the majority of perinatally exposed infants worldwide, including those enrolled in prevention of mother-to-child transmission programs, is hampered by lack of access to an HIV diagnosis in infancy. Dried blood spot collection from young infants for centralized HIV polymerase chain reaction (PCR) testing is attainable in low-resource settings, provided PCR methodology suitable for routine laboratory service is available. The accuracy of the Roche Amplicor HIV-1 DNA test version 1.5 (Branchburg, NJ) performed on dried blood spots collected prospectively on ordinary Whatman filter paper from a cohort of 300 6-week-old infants born to HIV-infected women in Johannesburg, South Africa, was assessed. Anonymous analysis of the blood spots using a unique DNA extraction procedure was performed in a routine diagnostic laboratory and the results compared with HIV DNA and RNA PCR liquid blood tests at age 6 weeks, and the HIV status of the infant. Dried blood spots were available for 288 infants (96%) of whom 25 (8.7%) were HIV infected. The Roche Amplicor assay yielded a sensitivity of 100% and a specificity of 99.6%. HIV DNA PCR tests on dried blood spots have the potential to improve health care delivery to HIV-affected children in low-resource settings right now.