A set of 17-point mutants of H-2Kd was produced as secreted single-chain MHC molecules in the yeast Kluyveromyces lactis. Using a library of 648 synthetic peptides displaying the Kd-specific motif, the repertoire of peptides selected by each mutant was compared by a two-dimensional analysis technique. When conserved residues of Kd were substituted by an alanine, no binding was observed. When polymorphic residues were changed, two outcomes were observed. For residues 95, 99, and 116, no binding was observed, implying that the side chains of these residues contribute directly to the interaction with the peptide. When positions 9, 45, 97, and 114 were changed to alanine, the repertoire of selected peptides was enlarged. Position 97 was also changed to all four possible residues found in natural variants of mouse MHC class I molecules. The repertoires of selected peptides were analyzed and appeared to be included one in another. Their dimension was inversely correlated with the size of the side-chain of residue 97. We conclude that during the course of evolution some polymorphic residues may have been selected for their capacity to reduce the size of the peptide repertoire by preventing certain peptides from binding.