Mouse monoclonal antibodies (mAbs) with the ability to inhibit thyrotropin (TSH) binding to the TSH receptor (TSHR) are useful tools to study TSH-TSHR interaction. The 3C3 mAb we produced was found to inhibit binding of TSH to human (h)TSHR but not to porcine (p)TSHR. Purified 3C3 immunoglobulin G (IgG) and its antibody-binding framgnet were prepared using standard methods and their ability to inhibit TSH binding to hTSHR or pTSHR was analyzed using a coated tube assay. The TSHR epitope reactive with 3C3 IgG was determined using Western blotting, ELISA based on peptides corresponding to the TSHR sequence, and the SPOT synthesis technique. RNA was isolated from 3C3 hybridoma cells and the mAb variable (V) region genes were sequenced and analyzed. 3C3 mAb had a 1x108 l/mol binding affinity to the hTSHR as assessed by Scatchard analysis. 3C3 reacted with the hTSHR region between amino acids (aa) 212-230, and two aa differences were found between the corresponding regions in the hTSHR and pTSHR. The light chain (LC) genes of 3C3 were derived from the Vk21 germ line (97.6% homology) and Jk2 genes. The heavy chain (HC) genes were from the V130 germ line (94.6% homology) combined with a D gene (not identified) and JH3 gene. The replacement/silent mutation ratios of 6.0 and 6.5 for the LC and the HC V regions, respectively, indicated that 3C3 underwent antigen-driven maturation. Mouse mAbs of this type should be useful in studying the interactions between the TSHR, TSH, and mAbs in more detail.