We have produced four monoclonal antibodies (mAbs), 34A, 49G, 11E7, and 12E3, which bind the human TSH receptor (hTSH-R) when expressed on a human thyroid cell line (GEJ), freshly dissociated human and murine thyroid cells, or Chinese hamster ovary cells stably transfected with the hTSH-R gene. These mAbs were obtained after immunization of DBA/1 mice with affinity-purified TSH-binding sites from GEJ cells. Biochemical studies, including sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, Western blot, and immunoprecipitation of solubilized GEJ cell membranes or human thyroid cells showed that most of the mAbs recognized two bands: one located at 46-48 kilodaltons and the other at 86-88 kilodaltons. Inhibition of [125I]hTSH binding to solubilized porcine membranes (TSH-receptor auto-antikörper assay) or Chinese hamster ovary cell membranes previously transfected with hTSH-R gene showed that mAb 34A recognizes the hTSH-binding site of both receptors. In contrast, mAbs 49G, 11E7, and 12E3 recognize a structure located near the hTSH-binding site. Lastly, the ability of these mAbs to stimulate murine thyroid function was investigated by measuring cAMP production and iodide accumulation. The 34A mAb, which fully competes with [125I]TSH for binding to hTSH-R, was able to induce both functions. Conversely, the 12E3 mAb, which was the least potent inhibitor of [125I]TSH binding to hTSH-R-transfected cells had no effect. A relationship was, therefore, established between the capacity of mAb to hTSH-R to inhibit [125I]hTSH binding and their ability to induce thyroid functions.