Certain major-histocompatibility-complex (MHC) loci are highly polymorphic, and the mechanism of maintenance of this polymorphism remains controversial. Recent studies of the pattern of nucleotide substitution at MHC loci have produced strong evidence that this polymorphism is maintained mainly by positive Darwinian selection that operates on the antigen recognition site (ARS) of the MHC molecule. The ARS of the class I MHC consists of three subregions: (1) the binding cleft, (2) T-cell-receptor-directed residues, and (3) outward-directed residues. Here we report that the rate of nonsynonymous nucleotide substitution is much higher in the binding cleft than in the other ARS subregions. Furthermore, nonsynonymous nucleotide substitutions that result in a change of residue side-chain charge occur significantly more frequently than expected by chance. We conclude that the main target of positive selection on the class I MHC molecules is the binding cleft of the ARS and that this selection acts primarily to promote diversity among alleles with respect to the pattern of residue side-chain charges (charge profile) in the binding cleft. These results provide additional support for the hypothesis that MHC polymorphism is maintained by overdominant selection relating to antigen-binding capacity and thus to disease resistance.