The great majority of insulin-specific T cell clones isolated from islets of NOD mice react with insulin peptide B-(9-23) (amino acids 9-23 of the insulin B chain). The T cell receptors of these clones contain diverse beta-chains but restricted alpha-chains. The dominant alpha-chain motif is a V alpha 13 segment (10 out of 13) combined to either a J alpha 45 or a J alpha 34 segment (eight out of 13). Furthermore, nine out of 10 of these V alpha 13 segments are a product of a novel NOD TCR V alpha gene which we have termed V alpha 13.3. Analysis of V alpha 13 transcripts from splenic cDNA libraries from the NOD, BALB/c and C57BL/6 mice revealed significant differences between strains. The NOD sequences contained both V alpha 13.1 and the novel V alpha 13.3. The BALB/c contained the previously reported V alpha 13.1 and V alpha 13.2, but not the V alpha 13.3 sequence identified in the NOD anti-insulin T cell clones. The C57BL/6 had V alpha 13.1 and V alpha 13.3 plus two additional novel sequences which we have termed V alpha 13.4 and V alpha 13.5. These V alpha 13 subfamily members differed by two to four amino acids in either the CDR1 region or adjoining the CDR2 region. The frequency of utilization of the different V alpha 13 subtypes varied dramatically between strains. In the NOD spleen, V alpha 13.3 was detected 79% of the time, compared to 21% for V alpha 13.1. In contrast, the C57BL/6 spleen contained only 7% of V alpha 13.3 sequences compared to the other subfamily members present (V alpha 13.1: 27%; V alpha 13.4: 56%; V alpha 13.5: 10%). MHC polymorphisms or other unknown selective pressures may contribute to these differences in V alpha 13 utilization. We hypothesize that the presence and frequent utilization of the V alpha 13.3 T cell receptor element is involved in targeting insulin B-(9-23) and may be related to diabetes susceptibility of NOD mice.