From the peripheral lymphocytes of a patient with Graves' disease, we established a T cell line using its reaction to a pool of 49 synthetic peptides corresponding to the entire human thyrotropin receptor (TSHR) sequence. This T cell line showed a specific response to the pool of peptides in a microproliferation assay (stimulation index: 4.8). Flow cytometry analysis revealed that the cell surface markers were CD4+ CD8-, T cell receptor (TcR) alpha beta+, and Tcr gamma delta-. To investigate T cell epitopes on TSHR, the T cell line reacted well against three groups: the N-terminal (amino acids 31-169) and C-terminal (338-420) regions of the extracellular domain and the N-terminal half (441-661) of the transmembrane domain of the receptor. This suggests a multiplicity of T cell epitopes on the TSHR, and was further supported by analysis of TcR gene expression in the cell line that showed the expression of 5 V alpha genes; V alpha-1, 2, 10, 20, and w25. In conclusion, the results of the present study indicated multiple T cell epitopes on the TSHR molecule including the transmembrane domain.