To examine the heterogeneity of autoantibodies to the human TSH receptor (hTSHR), we evaluated 20 sera from patients with Graves' disease for their recognition of prokaryotic (unglycosylated) and eukaryotic (insect cell glycosylated) recombinant hTSHR extracellular domain (ecd) in an unfolded (linear) and a folded (nonlinear) state. With the prokaryotic antigen, 12 (60%) bound folded hTSHR ecd monomer, 8 (40%) bound to the unfolded monomer, and 3 (15%) bound to a tetrameric species. Such binding to different hTSHR antigens was not mutually exclusive. In addition, 7 (35%) sera showed an apparently higher reactivity for the folded than the unfolded monomer. When reacted against the glycosylated insect cell hTSHR ecd, 9 (45%) sera recognized both the unfolded and folded monomer, and 5 (25%) recognized the tetrameric form. In all of our testing, 17 of the 20 sera (85%) bound to 1 or more of the recombinant hTSHR ecd antigens, and the recognition pattern appeared to be heterogeneous in at least 4 (20%) of the serum samples, with hTSHR antibodies recognizing linear, folded, and glycosylated hTSHR ecd monomers. We conclude, therefore, that patients with Graves' disease have autoantibodies that recognize multiple epitopes on the hTSHR ecd and that it is possible to classify them according to their recognition of linear, folded, and glycosylated products.