We evaluated a rapid membrane-based assay (HIV-CHEK) for detection of antibodies to HIV using 737 serum samples in Nairobi, Kenya. The rapid assay had a sensitivity of 96.3% and specificity of 99.8% when compared with enzyme-linked immunosorbent assay (ELISA) and Western blot assay. Results were similar using fresh or previously frozen serum samples, although the latter occasionally left debris on the assay device membrane yielding uninterpretable results. This rapid HIV assay may be of particular use in developing countries where laboratory resources are limited.