A new enzyme immunoassay (EIA), the Cobas Core Anti-HIV-1/HIV-2 EIA DAGS (also referred to as Roche DAGS), for the detection of antibodies to human immunodeficiency virus type 1 (HIV-1) and HIV-2 was evaluated in four centers. The assay is based on the double-antigen sandwich (DAGS) format, which enables the detection of all classes of antibodies. The antigens consist of recombinant proteins in their native conformation and of synthetic peptides. Of a total of 5,836 negative serum samples, including 95 samples likely to produce false reactivities, 6 were false positive, resulting in a specificity of 99.9%. None of 35 sera that were from noninfected individuals but contained p24-cross-reacting antibodies as revealed by Western blot (immunoblot) analysis were reactive by the Roche DAGS assay. In samples from individuals infected with HIV-1 group M (n = 499) and HIV-2 (n = 200), the sensitivity of the assay was 100%. Although containing antigens with sequences from subtype B only, the assay was also able to correctly identify with high optical density/cutoff ratios samples from subjects infected with HIV-1 subtype O (n = 10). In 17 of 19 seroconversion panels tested, the assay detected the presence of HIV-1 antibodies as early as another sandwich EIA. Eight of these panels were also analyzed by an indirect second-generation assay, which detected antibodies 2 to 10 days later than did the DAGS assay under evaluation. The excellent specificity and sensitivity of the new Cobas Core Anti-HIV-1/HIV-2 EIA DAGS are the result of the DAGS format as well as of the native, naturally folded form of the recombinant protein used as the gag antigen.