Major histocompatibility complex (MHC) class I molecules are cell-surface glycoproteins that bind peptides and present them to T cells. The formation of a peptide-MHC complex is the initial step in specific, T cell-mediated immune responses. But, unlike other receptor-ligand systems, peptides are essential for a stable conformation of the MHC proteins. To investigate the contribution of every amino acid of octapeptides to the stability and antigenic integrity of MHC proteins, complex octapeptide libraries with one defined amino acid and mixtures of 19 amino acids in the remaining seven positions were synthesized and tested for their capacity to stabilize the conformation of the mouse MHC class I molecule H-2Kb. Peptide transporter-deficient RMA-S cells were employed in this study. Amino acid preferences found for the eight sequence positions reveal constitutional, volumetric, and steric constraints that govern peptide selection by MHC molecules. The pattern of amino acid preferences indicates that the peptides behave as integral parts of the MHC proteins and follow rules established for the interrelationship of primary sequence and the conformation and stability of proteins in general.