We report a method for the purification and radioactive labeling of human TSH receptor (TSHR). The method is based on the construction of a fusion TSHR (TSHR–Xa–BIO) which consists of the N-terminal 725 amino acids of human TSHR linked to the 4-amino acid Xa protease cleavage site and the 87-amino acid C-terminal domain of the biotin carboxyl carrier protein subunit of Escherichia coli acetyl-CoA carboxylase (the C-terminal domain directs the efficient posttranslational biotinylation of the protein). TSHR–Xa–BIO was produced in HeLa cells using recombinant vaccinia virus. The expressed protein was fully functional and was biotinylated with an efficiency of about 90%. Streptavidin–agarose-immobilized TSHR–Xa–BIO was labeled with 125I using the chloramine T oxidation procedure and specifically eluted from the solid phase after cleavage with protease Xa. Isolated native radiochemically pure 125I-labeled TSHR specifically interacted with pathological autoantibodies in the sera of patients with Graves’ disease, and thus could be useful for the detection of these autoantibodies by immunoprecipitation analysis.