Myelin oligodendrocyte glycoprotein (MOG) is a major experimental autoimmune encephalomyelitis (EAE) antigen in H-2b mice and a potential autoantigen in multiple sclerosis. How well MOG peptides bind to MHC and how TCR recognize the peptide/MHC complex have important implications for thymic selection as well as T cell activation in the periphery. In this study, we have characterized amino acids in the MOG(38-51) peptide important for peptide binding to I-Ab, and for TCR recognition of the peptide/MHC complex. We found that the amino acids R41, F44, R46 and V47 constituted the major TCR contact residues, as alanine substitution at these positions abrogated T cell responses without decreasing their binding affinity to I-Ab. In addition, G38 and W39 were found to be minor TCR contact residues. Finally, substituting tyrosine for alanine at position 40 decreased binding to I-Ab by approximately 50% and prevented induction of T cell responses in C57BL/6J mice upon immunization. Thus, Y40 is the dominant MHC-binding residue of the MOG(38-51) peptide and most likely occupies the p1 pocket of I-Ab. Our results could be useful to design peptides with altered agretopes and epitopes of the MOG(38-51) peptide to study their therapeutic potential in the EAE model.