The non-obese diabetic (NOD) mouse develops both a spontaneous T-cell-mediated autoimmune insulitis and, in addition, a well characterized thyroiditis. We have examined the repertoire of murine T-cell receptor (TCR) variable (V) beta-chain genes used by intrathyroidal T cells with specific oligonucleotides that amplified 17 murine V beta gene families in cDNA samples prepared from intact NOD thyroid tissues. Normal NOD thyroid tissue contained only low levels of TCR V gene mRNA. In contrast, NOD mice with histologic thyroiditis showed the marked expression of up to 3 TCR V beta genes consistent with a restricted T-cell invasion. Sequencing of amplified TCR V beta cDNA showed that within each NOD thyroid sample at least one of the overexpressed V beta gene families was clonally expanded. However, the clonally expanded T-cell V gene family was not consistent in all animals. Even within the same TCR V beta gene families, various D and J segments had been rearranged with open reading frames and together with insertions and deletions gave no significant homology at the nucleotide or amino acid level. In summary, these data showed that the intrathyroidal T-cell infiltrate in NOD mice was markedly biased towards the use of a single, but variable, TCR V gene family within each animal. It also appeared that the choice of the TCR V beta chain determined the intrathyroidal infiltrative process rather than the choice of D and/or J regions. However, there was no consistent use of a single TCR V beta chain. As thyroiditis does not occur uniformly in apparently genetically homogeneous animals, reared under similar environmental conditions, it may not be surprising that different TCR V genes are involved in different animals.