Insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice results from a cell-mediated autoimmune process against pancreatic beta-cells. We have shown that beta-cell-cytotoxic CD8+ T cell clones can transfer IDDM to irradiated NOD mice if co-injected with nondiabetogenic CD4+ spleen T cells. To determine whether CTLs recruited to pancreatic islets recognize a restricted set of local Ags, we sequenced TCR-alpha and TCR-beta cDNA generated by anchor PCR from CD8+ CTL lines and clones derived from islets of 10 different NOD mice. These CTL lines were oligoclonal, but did not show skewed V alpha, V beta, J alpha, or J beta gene usage when compared with CD8+ spleen T cells. However, of the 26 different CTL-derived TCR-alpha sequences from all of these CTL lines and clones, 17 (65%) used one of three highly related, N region-encoded, CDR3 motifs. Motifs 1 and 2 (7 clonotypes each) contained a hydrophobic amino acid followed by Arg and a negatively charged or a polar residue (Asn or Gly), respectively. Motif 3 (3 clonotypes) was x-Arg-Gly. In 12 of these 17 rearrangements, the core sequence was followed by Tyr or Ser. By contrast, none of 31 different TCR-alpha rearrangements used by CD8+ spleen T cells encoded motifs 1 or 2, and only one encoded motif 3. Different TCR-beta rearrangements within individual lines also used homologous CDR3 sequences, but these sequences varied between lines. Skewed TCR-alpha-CDR3 usage by islet-derived CTLs was substantiated further by isolation of CTL clones transcribing highly homologous TCR-alpha, but different TCR-beta, rearrangements. These data suggest that CTLs recruited to pancreatic islets during spontaneous IDDM recognize a restricted set of beta-cell autoantigenic determinants.