We developed and evaluated two peptide-based immunoassays to confirm and discriminate between group M and group O HIV-1 infection. These assays are based on in vitro competition for antibody binding between M and O peptides. The first EIA is based on competition between group M and group O gp41 immunodominant domains and the second on competition between group O and group M V3 regions of gp120. Two panels of sera were used: the first consisted of 109 sera collected from 27 group O- and 92 group M-infected patients in whom the HIV isolates had been genotyped by sequencing or heteroduplex mobility assay. In this panel, the combination of the two assays correctly discriminated 106 samples (100% group O and 96.7% group M samples). The second panel, used for the field evaluation of the two assays, consisted of 157 samples from HIV-1-infected Cameroonian patients, 33 strains having been genotyped. The combination of the two techniques in a serogrouping algorithm discriminated 147 of these samples, 74 being HIV-1 group O and 73 group M. These results always correlated with genotyping results. The 10 sera that were not successfully classified by these assays were from early seroconverters. Altogether, the two assays clearly differentiated 263 of 276 (94.9%) samples in the two panels. On the basis of the genotyping results, the positive predictive value for group discrimination in the two panels was 100% for both GSEIA assays. Our peptide-blocking group-specific EIAs for differentiation and confirmation of HIV-1 group M and group O infection are complementary tools for epidemiological studies and surveillance of HIV-1 group O strain trafficking.