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To identify cost-effective testing strategies for HIV-1 and HIV-2 infections, we evaluated different combinations of tests on serum specimens from 1134 consecutive patients attending tuberculosis treatment centers in Abidjan, Cote d'Ivoire. Virus-specific whole-virus enzyme-linked immunosorbent assay (WVE), Western blot (WB) and synthetic peptide enzyme-linked immunosorbent assay (SPE) were used in sequential fashion to determine the true prevalence of infection; 27% were reactive to HIV-1, 5% to HIV-2, and 10% to both viruses. Of 239 specimens positive on WB for both HIV-1 and HIV-2, SPE diagnosed 38% as HIV-1-reactive and 16% as HIV-2-reactive, while 46% remained reactive to both viruses. Using WVE or one of two rapid (5–10 min) mixed (HIV-1 and HIV-2) antigen tests (RMATs) as a screening test, followed by SPE as a supplemental test, gave results with sensitivity of 97.3–99.2%, specificity of 99.5–99.7%, and positive predictive value for diagnosing HIV infection of 99.4–99.6%, with important savings in time and reagent costs. SPE allows more specific distinction between HIV-1 and HIV-2 infections than WB, and could replace it as a supplemental test in many settings. WB may be required for specimens reactive on screening tests but negative on SPE, until sensitivity of the SPE is further evaluated. A mixed antigen screening test followed by SPE seems to be an efficient testing strategy for diagnosing HIV-1 and HIV-2 infections.