The inner domain of the human immunodeficiency virus (HIV-1) gp120 glycoprotein has been proposed to mediate the noncovalent interaction with the gp41 transmembrane envelope glycoprotein. We used mutagenesis to investigate the functional importance of a conserved beta-sandwich located within the gp120 inner domain. Changes in aliphatic residues lining a hydrophobic groove on the surface of the beta-sandwich decreased the association of the gp120 and gp41 glycoproteins. Other changes in the base of the hydrophobic groove resulted in envelope glycoproteins that were structurally intact and able to bind receptors, but were inefficient in mediating either syncytium formation or virus entry. These results support a model in which the beta-sandwich in the gp120 inner domain contributes to gp120-gp41 contacts, thereby maintaining the integrity of the envelope glycoprotein complex and allowing adjustments in the gp120-gp41 interaction required for membrane fusion.