The advent of site-directed serology has provided a specific and simplified means of distinguishing antibody responses to the two types of human immunodeficiency virus (HIV) in an outbred population. Remarkably, human sera containing HIV antibodies and simian sera containing simian immunodeficiency virus (SIV) antibodies had a very narrow amino acid dependence in the 23 residue long peptide, 582Ala-604Cys, used as antigen. A single dominating antigenic site was demonstrated in the C-terminal part of the peptide--596Trp-602Gln for HIV-2 and SIV antibodies and 597Gly-602Leu for HIV-1. A minor fraction of HIV-1 antibodies also reacted with a second site, 588Lys and 589Asp. These data define the precise amino acid dependence of a uniquely immunogenic site in the folded transmembrane protein of HIV and facilitate the optimizing of peptide antigens for site-directed serology.