The N-terminal peptide Ac1-11 of myelin basic protein induces experimental autoimmune encephalomyelitis in H-2(u) and (H-2(u) x H-2(s)) mice but does not in H-2(s) mice. Ac1-11 binds weakly to the class II major histocompatibility complex (MHC) molecule I-Au but not at all to I-As. We have studied the interaction of Ac1-11 and I-Au as a model system for therapeutic intervention in the autoimmune response seen in experimental autoimmune encephalomyelitis. Two polymorphic residues that differ between I-Au and I-As, Y26beta and T28beta, and one conserved residue, E74beta, confer specific binding of Ac1-11 to I-Au. A fourth residue, R70beta in I-Au, affects both peptide binding and T cell recognition. These results are consistent with a model that places arginine at position five of Ac1-11 in pockets 4 and 7 of the MHC groove, which is formed in part by residues 26, 28, 70, and 74 of Abetau and places lysine at position four of Ac1-11, previously shown to be a major MHC contact, in hydrophobic pocket 6. The data indicate that the primary region of I-Au that confers specific binding of Ac1-11 lies in the center of the peptide binding groove rather than in the region that contacts the N terminus of the peptide, as has been shown for HLA DR and the homologous I-E molecules.