The first step in infection of human mononuclear cells with HIV involves the high affinity binding of the viral envelope glycoprotein, gp120, to the cell-surface receptor, CD4. To gain a better understanding of the molecular basis of this interaction, we have analyzed the ability of gp120 to bind to a panel of 40 mutant CD4 proteins containing single or double amino acid substitutions. In addition, the binding of several anti-CD4 mAb to the mutant CD4 proteins was measured. These mAb were chosen on the basis of the previous demonstration that they bind to epitopes in CD4 adjacent to the gp120-binding site. This analysis permits discrimination between mutations that probably cause localized conformational changes and those that alter residues likely to make direct contact with gp120 and with the mAb. Our results indicate that gp120 from two different strains of HIV binds to a larger region of the CD4 protein than previously described. The data has also been used to map the epitopes of mAb previously identified as anti-idiotype vaccine tes. The results have important implications for the development of CD4-based therapies for AIDS.