The diagnosis of HIV infection is usually determined using serological methods. Positive results in a screening test should be confirmed with more specific supplementary assays, and the Western blot (WB) is the most frequently used. However, problems of WB, particularly in regard to the significance of indeterminate results, still remain. We evaluate a new recombinant HIV-1/2 antigen-based immunoblot (RIBA) as confirmatory assay in a panel of 190 clinical samples. The RIBA was much more sensitive and specific than WB. In particular, the test allows for a significant reduction in the number of indeterminate results and for more accurate distinction between HIV-1 and/or HIV-2 infections. Thus, these results make tests such as the RIBA instead of WB particularly attractive for validating anti-HIV EIA reactivity.