A human anti-HIV monoclonal antibody (mAb), 2909, selected on the basis of its potent neutralizing activity against HIV-1SF162, recognizes a complex epitope V2/V3 present on intact virions but not on soluble gp120. To confirm the quaternary nature of the epitope, 2909 binding was tested against the pseudovirus SF162 wild type (WT) expressing trimers and/or an SF162 mutant expressing monomeric envelope proteins. The construction of the SF162 mutant was made by an alanine substitution of nine hydrophobic residues in the N-terminal heptad repeat region of gp41 molecules that failed to form trimers on the virus surface. Monoclonal Ab 2909 bound only to SF162 WT virions and transfected cells as determined by immunoprecipitation and flow cytometry, respectively, but showed no reactivity to the SF162 mutant expressing monomeric gp120. The data provide further evidence for the existence of a unique quaternary epitope V2/V3 on the surface of unliganded virus.