Through mutagenesis, we identified a single high-affinity binding site for gp120 on the human CD4 protein. This site is localized in the V1 domain within residues 41 to 55. The collection of mutants was also used to define the epitopes for 55 anti-CD4 monoclonal antibodies. The locations of these epitopes are consistent with a V kappa-like structure for the V1 domain. In the context of this structure, the gp120 binding site encompasses the small CDR2 loop. Through deletion mutagenesis at the termini of the V1 domain, we further defined the minimal region required to retain high-affinity binding to gp120. Short deletions at both termini disrupt binding to gp120 and recognition by conformation-sensitive anti-CD4 monoclonal antibodies. We conclude that amino acids at both the amino and carboxy termini are critical to the conformation of the V1 domain and, in particular, to the integrity of the gp120 binding site.