The aim of the study was to evaluate a new ELISA for detection of HIV-1, HIV-2 and HIV-1 subtype 0 (HIV-0) antibodies. The assay format is based on the antigen sandwich principle. To enable specific detection of HIV-0 antibodies, in addition to HIV-1 and HIV-2 antigens HIV-0 antigen is used for coating the solid phase and for the conjugate. The results show that all 12 HIV-0 samples tested were detected with a high degree of reactivity, as were all the 1,144 anti-HIV-1 and 424 anti-HIV-2 positive samples. The capacity of the test to enable early detection of seroconversions is equivalent to that of other sandwich ELISAs. The specificity of the assay was determined to be 99.89/99.94% (initial/after retest) using 58,366 samples, which is superior to the other ELISAs used for comparison. Even with difficult samples (i.e. samples of African origin, samples known to cause false-positive reactivity in different ELISAs, or samples containing potential interference factors) there were very few false-positive reactions. Therefore, the new assay is well suited for screening blood donations as well as for evaluating samples from patients of different geographic origin.