To date, candidate HIV-1 vaccines that have been tested in clinical trials have failed to induce broadly neutralizing activities and/or antibodies that inhibit infection by primary isolates of HIV-1. We recently identified a conserved caveolin-1 binding motif, WNNMTWMQW, in the ectodomain of HIV-1 transmembrane envelope glycoprotein gp41. We designed the synthetic CBD1 peptide SLEQIWNNMTWMQWDK, corresponding to the consensus caveolin-1 binding domain (CBD) in gp41, and showed that it elicits in rabbits the production of antibodies that inhibit infection of primary CD4(+) T lymphocytes by various primary HIV-1 isolates. Although a conserved and highly homologous caveolin-1 binding motif is present in the transmembrane envelope glycoprotein of different HIV-2 isolates, anti-CBD1 immune sera do not inhibit HIV-2 infection. Here we show that anti-CBD1 antibodies are directed against the conserved caveolin-1 binding motif WNNMTWMQW in the CBD1 epitope. In spite of this, anti-CBD1 antibodies do not react with the CBD2 peptide SLTPDWNNMTWQEWER, corresponding to the potential consensus caveolin-1 binding domain in HIV-2. The presence of a conserved proline residue upstream of the caveolin-1 binding motif in CBD2 might affect the presentation of this motif, and thus account for the lack of reactivity of the immune sera. Anti-CBD1 antibodies therefore appear to be directed against a conformational epitope mimicked by the synthetic CBD1 peptide. In accordance with this, anti-CBD1 immune sera react with the native but not denatured gp41. The reactivity of anti-CBD1 immune sera with a highly conserved conformational epitope could explain the broad inhibitory activity of such antipeptide antibodies against HIV-1 isolates of various clades.