Previous studies by us and others showed that HIV-1 gp41 by the immunosuppressive domain (amino acids 583-599) could bind to human T, B and monocytic cells. Here, we characterize a region (amino acids 641-685) of HIV-1 gp41 that may be another site for gp41 binding. Besides peptides of the gp41 immunosuppressive domain, peptides of a second region (amino acids 641-685) could also inhibit rsgp41 (amino acids 539-684) binding to human cell lines U937 (monocytic cell line) and Raji (B cell line). An epitope (amino acids 669-674, ELDKWA) within this region is recognized by human monoclonal antibody 2F5. 2F5 shows broad-spectrum neutralizing capacity against HIV-1 laboratory strains and primary isolates (J. Virol. 1994, 68: 4031-4034; AIDS Res. Hum. Retrovir. 1994, 10: 1651-1658). We demonstrated by flow cytometry that mAb 2F5 could block gp41-binding to human cell lines H9 (T cells), Raji and U937, but the control mAb 4E10 that recognizes another region of gp41 (amino acids 686-728) and can not inhibit HIV infection, did not block this binding. These results indicate that the region (amino acids 641-685) of HIV-1 gp41 is the second site by which gp41 interacts with human cells, and suggest that the neutralization of HIV-1 infection by mAb 2F5 may be based on the fact that it blocks interaction of gp41 on virions with the gp41 binding molecules on human cells.