T cells isolated from islets of non-obese diabetic (NOD) mice are enriched for insulin-reactive cells. The great majority of these T cells recognize insulin B chain peptide (B:9–23). B:9–23 reactive T cell clones are diabetogenic and show a dramatic TCR α -chain restriction (predominant AV13S3). We have studied the reactivity of five different B:9–23 reactive T cell clones to truncated peptides and alanine substituted analogues of B:9–23. Amongst these AV13S3 T cell clones, one reacted with peptide B:9–16 and four with B:13–23. The two peptides have in common only four amino acids (B:13–16; EALY). Having defined minimal peptide epitopes, we evaluated a mutant insulin sequence (B:13 glutamine) which retains metabolic activity. As predicted, this single amino acid change abrogated T cell reactivity. In addition, we have created a modified I-A^g7gene with the B:9–23 peptide covalently linked to I-A^g7. Antigen presenting cells transfected with this construct were excellent presenting cells for all clones studied. The definition of dual peptide motifs and creation of bioactive covalent I-A^g7-B:9–23 should facilitate studies of the pathogenic significance and antigen recognition by B:9–23 reactive diabetogenic T cells.