Patients with Graves' disease have autoantibodies that bind to the TSH receptor and stimulate the thyroid, leading to hyperthyroidism. Earlier studies have shown that the ectodomain of the glycosylated human TSH receptor contains epitopes that could adsorb these pathogenic antibodies. Further studies with mutated cDNAs, chimeric proteins, peptides, and antipeptide antibodies suggested that alterations in the conformation of the protein could lead to loss of reactivity, and that thyroid-stimulating antibodies interact with the N-terminal region of the TSH receptor. Although many of these studies provided valuable insights, they were somewhat inconclusive due to limitations inherent to each of the approaches. In an attempt to further define regions within the TSH receptor with which thyroid-stimulating antibodies interact, we expressed seven recombinant TSH receptor fragments in insect cells and tested them for their ability to neutralize TSH binding inhibitory Igs and thyroid-stimulating antibody activity in the sera of patients with Graves' disease. The fragments containing amino acids 22-305 were able to neutralize the TSH binding inhibitory Ig activity, whereas a fragment containing amino acids 54-254 was able to neutralize the thyroid-stimulating antibodies. Fragments containing additional amino acids, flanking residues 54-254, failed to neutralize the thyroid-stimulating antibody activity, suggesting that thyroid-stimulating antibody epitopes are masked. Our studies show that thyroid autoantibodies, with different functional properties, bind to distinct conformational epitopes on the TSH receptor.