Several negatively charged amino acids of the human immunodeficiency virus type 1 (HIV-1) gp120 glycoprotein have been implicated in binding to the CD4 viral receptor. The CD4 region implicated in binding gp120 consists of a hydrophobic ridge protruding from a positively charged surface. To examine whether any of the surface charges on CD4 might contribute to gp120 binding, several amino acids near the CD4 regions previously implicated in gp120 binding were altered. Of the charged amino acids in the C'' and D strands of the amino-terminal domain of CD4, alteration of only two, lysine 46 and arginine 59, dramatically disrupted ability to bind gp120. In the three-dimensional structure of CD4, these two basic amino acids are located proximal to phenylalanine 43, which we confirmed to be important for gp120 binding. By contrast, we could not confirm the observations that alteration of residues in the F strand (CDR3-like region) of CD4 significantly affected gp120-binding ability. These results support a model of the gp120-binding site that is dependent on discontinuous amino acids but spatially limited.