Abstract. Epitope mapping of hTSH was carried out using 19 monoclonal antibodies prepared with hTSH or its β-subunit as antigen. The affinity constants of the monoclonal antibodies ranged from 9.6 × 107 to 5.7 × 109 mol/l for hTSH. The binding activities of monoclonal antibodies were maintained or in some cases rather enhanced after removal of the sugar moeity of the subunits of hTSH, and completely diminished after reduction of intramolecular S-S bonds in the subunits of hTSH. Ten monoclonal antibodies recognized the epitopes on hTSH (α:β subunit combined form) and on free α-subunit form. Eight other antibodies recognized the epitopes on free/or combined form of β-subunit, all of which did not recognize any other human glycoprotein hormones. The monoclonal antibodies directed against the α-subunit could bind also other human glycoprotein hormones to a varying extent. On the basis of results from competitive binding studies, the antibodies directed against α-subunit and those against β-subunit were each classified into five subgroups recognizing different antigenic determinants. The remaining one antibody recognized an epitope expressed only by hTSH and not by the free subunits. In addition, a positive cooperativity on the binding of hTSH was observed between monoclonal antibodies directed towards a particular epitope on the α-subunit and those towards an epitope on the β-subunit. From these data, two-dimensional map of epitopes on hTSH was constructed. The epitopes on each subunit were found to form a cluster with complicated overlapping, suggesting a highly conformational structure.