The characteristics of autoantibodies reactive with bovine (b) TSH were examined in the sera of six patients with Graves' disease selected on the basis of highly negative values in the TSH receptor assay. Test sera were incubated with other125I-labeled pituitary glycoprotein hormones and their isolated subunits (αandβ) [human (h) TSH, bTSH, porcine (p) TSH. pFSH, bFSH, bLH and equine (e) chorionic gonadotropin (CG)] (purity was confirmed by gel-filtration on Sephadex G-100 and SDS-PAGE), and the antibody bound fraction was precipitated by the addition of anti-human gamma-globulin (goat). Almost all sera showed detectable binding to bTSH, pTSH, pFSH, pTSH-α, bFSH-α, bLH-α, but not to hTSH, hTSH-α, hTSH-β, hFSH, hLH. hCG, pTSH-β, bLH-β, eCG-α. Exceptions were very low binding to bLH-βby one serum and to pTSH-βby two sera. The level of binding (B/T%) of the patients' sera to pTSH-α, bFSH-α, and bLH-α, was 3.0–17.7%, 2.6–45.3% and 2.2–39.0%. respectively; that of sera from normal healthy adults was 1.9±0.3%, 0.8±0.2% and 0.9±0.2% (mean±SD), respectively. These results indicate that the TSH antibodies recognize mainly an epitope in theαsubunit of bovine and procine pituitary glycoprotein hormones (TSH, FSH, LH).