In a panel of 13 mouse monoclonal antibodies generated against native (nondenatured) human thyroid peroxidase (TPO), only 1 (monoclonal antibody 47) recognized TPO protein fragments expressed in a human TPO cDNA sublibrary. Determination of the nucleotide sequences of 18 clones recognized by monoclonal antibody 47 localized its epitope to 9 amino acids (residues 713-721) in the human TPO protein. On Western blot analysis, only TPO monoclonal antibody 47 recognized the 933-amino acid TPO molecule after denaturation and reduction of the latter, supporting the concept that the major part of the epitope is represented by a continuous portion of the TPO sequence. The binding of TPO monoclonal antibody 47 to native TPO is inhibited by immunoglobulin G in the serum of patients with autoimmune thyroid disease. The epitope for monoclonal antibody 47 defined in the present study is, therefore, part of or in the vicinity of an epitope for autoimmune thyroid disease-associated TPO antibodies.