The HIV-1 gp41 (glycoprotein 41) core plays a critical role in fusion between the viral and target cell membranes. We previously identified a gp41 core-binding motif, HXXNPF, by screening the phage display peptide libraries. In the present study, we elucidated the mechanism of action of HXXNPF motif-containing molecules of different sizes, including the phage clone L7.8 (a selected positive phage clone), L7.8-g3p* (a 10-kDa fragment of the gene 3 protein) and JCH-4 (a peptide containing 13 residues of L7.8-g3p*), regarding their respective binding abilities to the six-helix bundle and inhibition on syncytium formation at different temperatures. We found that all of the HXXNPF motif-containing molecules could bind to the gp41 core, and that their binding sites may be located in the N-helix domain. L7.8-g3p* and JCH-4 effectively inhibited HIV-1 Env (envelope glycoprotein)-mediated syncytium formation at 37 degrees C, while the phage clone L7.8 showed no inhibition under the same conditions. However, at suboptimal temperature (31.5 degrees C), all of these HXXNPF motif-containing molecules were capable of inhibiting syncytium formation. These results suggest that these HXXNPF motif-containing molecules mainly bind to the gp41 core and stop the fusion process mediated by the fusion-active core, resulting in inhibition of HIV-1 fusion and entry. The HXXNPF motif-containing molecules may be used as probes for studying the role of the HIV-1 gp41 core in the late stage of the membrane-fusion process.