There has been considerable interest in the possible restriction of the TCR repertoire in autoimmune disorders, because it would have important therapeutic implications. Using ribonucleic acid derived from matched peripheral blood lymphocytes (PBL), intrathyroidal lymphocytes (ITL), and CD4- and CD8-selected ITL from three patients with Hashimoto's thyroiditis (HT), we carried out reverse transcription-PCR analysis of TCR V alpha family usage. No evidence was found for V alpha family restriction in the PBL, ITL, CD4-selected ITL, or CD8-selected ITL. However, restriction was frequent in the CD8-selected ITL after denaturation/reannealing of the PCR products followed by nondenaturing PAGE; similar restriction was uncommon in PBL, CD8-selected PBL, ITL, or CD4-selected ITL. V alpha 3 and V alpha 6 TCR chains from CD8-selected ITL bands from one patient were cloned and sequenced. There was marked sequence restriction, particularly within the ITL V alpha 6 TCR chains, in which 14 of 15 homoduplex band sequences used the J4 segment and had an identical V/N/J junction amino acid (but not nucleotide) sequence. Sequence restriction was not detected in matched CD8-selected PBL material. These data show that there is a marked restriction of V alpha chain usage in the CD8+ (but not CD4+) T cells in the Hashimoto's thyroid, with clonal expansion of some sequences.