We have investigated whether peptides representing the HIV-1 principal neutralization domain (V3) can be used as antigens in antibody-binding assays to predict the genotypes of the subjects' virus. Serum samples collected from HIV-1-infected subjects from the four WHO-sponsored vaccine evaluation sites (Uganda, Rwanda, Thailand, and Brazil) were characterized by antibody binding to a panel of synthetic V3 peptides that were derived from the consensus sequences of the V3 region of the HIV-1 subgroups according to the env phylogenetic analysis (A-E). An indirect V3 peptide-binding assay was used for primary screening, and a V3 peptide antigen-limiting ELISA was then used as a secondary assay to discriminate cross-reactivity if the screening assay was equivocal. In general, V3 peptide serology could predict HIV-1 genotypes. In sera for which the genotype of the virus was known, peptide assays could predict the correct genotype in approximately 90% of cases for genotypes A, B, C, and E; Ugandan sera of genotype D were more broadly reactive. There was considerable serological cross-reactivity between some HIV-1 genotypes, in particular between A and C, and, to a lesser extent, B and D subtypes. Owing to polymorphism at the crown of the V3 loop, an additional B peptide (B') was required to type Brazilian B genotype sera. These simple assays may help facilitate the determination and distribution of HIV-1 genotypes circulating in populations.