Rapid HIV assays have recently been shown to have important applications for various testing situations, including early identification of infected individuals, to allow intervention strategies in a clinically relevant time frame. A rapid, lateral flow, HIV-1/2/O assay was evaluated using 2,000 serum or plasma samples from various risk groups and geographic locations, including HIV-1 and HIV-2 positive sera from five countries. Two U.S. Food and Drug Administration (FDA)-licensed screening assays and a FDA-licensed confirmatory assay were used as reference tests. The rapid assay exhibited a near-perfect sensitivity (99.2%) and an excellent specificity (99.9%). Moreover, its analytical sensitivity was found to be better than most FDA-licensed enzyme-linked immunosorbent assays (ELISAs), detecting infection at the same time as the most sensitive ELISA in two of five seroconversion panels, and at the same time or earlier than four of five ELISAs in all five panels. We conclude that this rapid assay is a suitable test for the detection of HIV infection that could be particularly useful in developing countries where facilities may not support the use of instrumentation.