We have used cells infected with the HIV-1 molecular clone HX10 to study the binding of monoclonal antibodies (mAbs) to different epitopes within the extracellular domain of the HIV-1 transmembrane glycoprotein gp41. Gp41 mAb binding to the infected cells at 4 degrees was variable but weaker than the binding of an anti-gp120/V3 loop mAb and increased substantially for three of the gp41 antibodies at 37 degrees. Treatment of the cells with soluble CD4 (sCD4) at 37 degrees increased gp41 mAb binding to epitopes spanning residues 521-663, implying that these regions had probably been masked by gp120, which following interaction with sCD4 had subsequently dissociated from gp41. By contrast, the binding of a mAb to residues 662-667 which form a neutralization epitope was reduced by sCD4 binding. Another region which has been described as containing a neutralization epitope spans residues 725-750. MAbs to this region bound equally well to noninfected and HIV-infected cells, and binding was not increased in the presence of sCD4. These data strongly imply that this epitope is not exposed on the external surface of the membrane, a finding in accord with the proposed cytoplasmic localization of this region.