BACKGROUND Graves' disease (GD) is characterized by autoimmunity to the TSH receptor (TSHR). OBJECTIVE We sought to identify T cell epitopes in TSHR that initiate this immune response and their interaction with human histocompatibility leukocyte antigen (HLA) molecules predisposing to GD. METHODS We examined the affinity of 31 overlapping peptides spanning the TSHR extracellular domain for binding in vitro to five purified HLA-DR molecules; DRB1*0101 (DR1), DRB1*1501 (DR2), DRB1*0301 (DR3), DRB1*1101 (DR5), and DRB1*0701 (DR7). We scanned the TSHR extracellular domain using a T cell epitope-mapping algorithm, EpiMatrix. We compared these results with clinical studies of GD patients measuring in vitro T cell responses to the peptides. METHODS The study was conducted at a university laboratory. METHODS Patients included 200 serial adult clinic patients with GD. METHODS There were no interventions. METHODS Binding affinity of epitopes, predicted affinity, and reported T cell stimulation data were measured. RESULTS Most peptides bound with intermediate or high affinity to one or more HLA-DR molecule. Peptides binding to HLA-DR3 and HLA-DR5, which predispose to GD, exhibited moderate binding affinities overall, whereas most peptides binding to GD-protective HLA-DR7 bound with high affinity. These differences may relate to T cell selection in the thymus. Binding affinity of peptides correlated strongly with EpiMatrix-predicted affinity for HLA-DRB1*0101, DRB1*1501, DR3, and DRB1*0701 but not HLA-DR5. Average IC(50) values correlated significantly with clinical T cell stimulation data. CONCLUSIONS Three different methods for identifying immunogenic peptides did not provide a uniform picture of important TSHR epitopes. However, peptide 132-150 (GIFNTGLKMFPDLTKVYST) was identified by three methods as an important epitope in GD; the possible importance of peptides 145-163, 158-176, 207-222, 248-263, 272-291, and 343-362 was also identified.