We have analyzed protein folding and disulfide bond formation in the extracellular domain of the human TSH receptor (hTSHR-ecd) expressed in Escherichia coli. This domain, which begins at the amino-terminus and ends at residue 415, is a major autoantigen in human autoimmune thyroid disease. Refolding of reduced and denatured hTSHR-ecd occurred in polyacrylamide gels treated with 0.25 M KCl for visualization of protein bands. Under conditions of partial renaturation, at least three forms of the hTSHR-ecd were resolved by reelectrophoresis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis: 1) unfolded monomers, 2) folded monomers, and 3) tetramers. Disulfide bond formation was implicated in both folding and tetramerization, as reduction of these forms produced only unfolded monomers. A natural variant of the hTSHR (v1.3), sharing 231 N-terminal amino acids with hTSHR-ecd, formed folded monomers and dimers on renaturation, but not tetramers, implicating one or more of the five cysteine residues residing between positions 231-415 in the association of dimers into tetramers. Binding of three different sources of hTSHR antibodies to these various forms of the hTSHR-ecd was assessed by immunoblotting using: 1) murine monoclonal antibodies (MAbs) generated against hTSHR-ecd, 2) rabbit polyclonal antisera generated against overlapping synthetic peptides spanning residues 37-71 of the hTSHR-ecd, and 3) human immunoglobulin G from patients with Graves' disease and detectable hTSHR-Ab. One of the MAbs, shown to recognize residues 21-35, and the rabbit polyclonal antibodies bound to all three forms of the hTSHR-ecd. Some of the hTSHR autoantibodies bound predominantly to the monomeric forms of the hTSHR, but autoantibodies were also identified that recognized tetrameric hTSHR-ecd. These data demonstrate that hTSHR-Abs recognize differing nonlinear and linear epitopes in the hTSHR-ecd and provide a methodology that should be useful for further defining the structural requirements for folding of this functionally and immunologically important domain of the hTSHR.