The specificity of peptide binding to MHC molecules is defined by binding motifs composed of several relatively conserved anchor positions. The peptide binding motifs of murine MHC class II I-A molecules are functionally important but poorly characterized. Here we use peptide binding studies and isolation of naturally presented peptides to characterize the peptide binding motif of the MHC class II I-A molecule, RT1.B¹, a molecule that is involved in experimental autoimmunity in the Lewis rat. We now report that, similar to other class II motifs, the RT1.B¹ motif consists of a nonamer sequence with four major anchor positions (P1, P4, P6 and P9). Residues at P4 and P9, rather than at P1, appeared to be particularly important for binding. Negatively charged residues were favored at P9, consistent with the presence of a serine at position 57 of the RT1.B¹β) chain. This RT1.B¹ motif could be observed in the dominant autoantigenic T cell epitopes mapped previously in the Lewis rat. These results highlight a general similarity and some important differences in the organization of MHC class II peptide binding motifs. The reported RT1.B¹ motif should facilitate the prediction and design of T cell epitopes for the induction and control of experimental autoimmune diseases in Lewis rat models.