The class II molecules of the MHC bind processed Ag fragments (peptides) for presentation to T cells, but the role of individual MHC residues in binding these peptides has not been entirely defined. A panel of 27 mutant I-Ak transfectants was analyzed for the capacity to bind 2 unrelated peptides. The main peptides examined were hen egg lysozyme residues 48-62 and heat shock protein (hsp70) to residues 28-41. Alanine substitutions of sites in the alpha-helical region of the I-Ak alpha-chain altered the ability of this class II protein to bind both peptides. Of the 27 substitutions tested, nine caused a decrease in peptide binding while only three caused an increase in peptide binding. The stabilities of these altered I-Ak-peptide complexes were also examined on SDS-Page. Complexes with lowered stabilities were observed after only four substitutions, and in all four cases this loss of stability was accompanied by a loss in hen egg lysozyme or hsp70 peptide-binding ability. Further, three of these residues lie in the short extended strand at the N terminus of the alpha-helix of the alpha 1 domain, suggesting that this region of I-Ak molecule may be critical for the formation of stable peptide-MHC complexes.