Formation of the six-helix bundle (6-HB) core between the N- and C-terminal heptad repeats (NHR and CHR) regions of the HIV-1 envelope glycoprotein (Env) transmembrane subunit gp41 is a critical step during the process of virus and target cell membrane fusion. In the present study, we generated a panel of five monoclonal antibodies (mAbs) which specifically recognized the HIV-1 gp41 6-HB formed by the NHR-peptide N36 and CHR-peptide C34 mixture, but did not react with the isolated peptides N36 and C34. These mAbs did not block the HIV-1 Env-mediated cell-cell fusion at physiological temperature (37 degrees C), but inhibited the HIV-1 Env-mediated cell-cell fusion at suboptimal temperature (31.5 degrees C), under which condition the fusion process is slowed down and the viral 6-HB becomes accessible. The fusion inhibitory activity of the mAbs is correlated with their binding affinity with the 6-HB core. By screening 24 6-HB variants with single mutations at the b, c, and f positions in the helical wheels, we found that the critical binding sites of these mAbs were localized in the N-terminal region of the NHR and the C-terminal region of the CHR. These sites may serve as targets for design of small molecule HIV fusion inhibitors, e.g., organic compounds, peptides, and low molecular weight proteins.