Western blot (WB) is the most widely used serological confirmatory test of ELISA and rapid simple tests (RST) to detect infection with HIV. WB tests, however, are expensive, time-consuming, and have technical disadvantages. The authors therefore conducted a study to identify cost-efficient alternative strategies for HIV-antibody screening, confirmation, and discrimination of HIV-1 and HIV-2 infections in a HIV-1 and HIV-2 prevalent area. 1110 consecutively collected blood sera from Guinea-Bissau were included in the first phase of the study, of which 198 (17.8%) were known to be HIV-seropositive; 52 with HIV-1, 120 with HIV-2, and 26 being HIV-1/HIV-2 dually reactive. 95 selected HIV-positive specimens were included for study of sensitivity and cross-reactivity between HIV-1 and HIV-2, with WB used to confirm specimen reactivity. All specimens were screened by Enzygnost ELISA and Capillus RST, with reactive samples further tested by the following assays for confirmation: UBI ELISA, Innotest ELISA, Recombigen RST, Multispot RST, and Immunocomb RST. The confirmatory RST, Wellcozyme Recombinant HIV-1 ELISA, PEPTI-LAV, and INNO-LIA were also used to study differentiation between HIV-1 and HIV-2. All assays were 100% sensitive. The specificities of the screening assays at initial and repeated testing were 98.0% and 99.7%, respectively, for Enzygnost and 99.8% and 99.9%, respectively, for Capillus. Various combinations of 2-3 assays yielded specificities of 99.2-100%. Screening with Enzygnost ELISA and confirmation and differentiation between HIV-1 and HIV-2 with Capillus RST and Multispot RST was adopted for routine use at Guinea-Bissau's National Public Health Laboratory. A field trial of the approach conducted in 1996 involving 1501 sera found a sensitivity and specificity comparable to ELISA and WB.