The identification and epitope mapping of broadly neutralizing anti-human immunodeficiency virus type 1 (HIV-1) antibodies (Abs) is important for vaccine design, but, despite much effort, very few such Abs have been forthcoming. Only one broadly neutralizing anti-gp41 monoclonal Ab (MAb), 2F5, has been described. Here we report on two MAbs that recognize a region immediately C-terminal of the 2F5 epitope. Both MAbs were generated from HIV-1-seropositive donors, one (Z13) from an antibody phage display library, and one (4E10) as a hybridoma. Both MAbs recognize a predominantly linear and relatively conserved epitope, compete with each other for binding to synthetic peptide derived from gp41, and bind to HIV-1 MN virions. By flow cytometry, these MAbs appear to bind relatively weakly to infected cells and this binding is not perturbed by pretreatment of the infected cells with soluble CD4. Despite the apparent linear nature of the epitopes of Z13 and 4E10, denaturation of recombinant envelope protein reduces the binding of these MAbs, suggesting some conformational requirements for full epitope expression. Most significantly, Z13 and 4E10 are able to neutralize selected primary isolates from diverse subtypes of HIV-1 (e.g., subtypes B, C, and E). The results suggest that a rather extensive region of gp41 close to the transmembrane domain is accessible to neutralizing Abs and could form a useful target for vaccine design.Eliciting broadly neutralizing antibodies (Abs) to human immunodeficiency virus (HIV-1) is a major goal of vaccine research but one that has proved extraordinarily elusive (8,11,77). This probably reflects the low antigenicity and immunogenicity of the HIV-1 envelope spike and most especially of relatively conserved regions of the spike. It is clear that much of the protein surface of the gp120 and gp41 protein molecules in the heterotrimeric envelope spike (gp120 3 -gp41 3 ) is directly or indirectly occluded from Ab binding by protein-protein interaction. Thus, for example, extensive surfaces on gp41 appear to be involved in interaction with other gp41 molecules and with gp120 (62, 63). Reciprocally, a portion of the surface of gp120 is occluded by the interaction with gp41 and by trimer formation (28,32,75,76). The relatively low immunogenicity of HIV-1 envelope trimers is also inferred from the low titers of neutralizing Abs, particularly cross-isolate neutralizing Abs, elicited during natural infection (31,39,40). This follows since a good correlation has been established between Ab neutralization and binding to envelope spikes, at least for T-cell-line-adapted viruses (50, 57, 61), suggesting that the deficit in neutralization originates from a deficit in spike binding. Low immunogenicity presumably arises, at least in part, from the weakly stimulating properties of the exposed regions of the envelope trimer. These include extensive regions of carbohydrate. A caveat here is that one cannot generally be sure of the eliciting antigen: Abs reactive with the trimer may have been elicited by other...