To define the autoantibody epitopes in amino acids 513-633 of thyroid peroxidase (TPO), a region frequently recognized in thyroiditis, cDNA sequences coding for peptide fragments of this region were amplified and ligated into pMalcRI and pGEX vectors for expression as recombinant fusion proteins. Western blots and enzyme-linked immunosorbent assay were then used to examine the reactivity in sera from 45 Hashimoto's and 47 Graves' disease patients. Two autoantibody epitopes within TPO amino acids 589-633 were identified; 16 of 35 patients reactive to TPO513-633 recognized the epitope of TPO592-613, while 6 patients recognized the epitope of TPO607-633. Eleven other patients with thyroiditis and two with Graves' disease recognized only the whole 589-633 fragment, and this response accounted for the Hashimoto's disease specificity. An amino acid sequence comparison of TPO592-613 with analogous regions of other peroxidase enzymes revealed significant differences in this area, and the substitution of even a single amino acid in one of the epitopes markedly decreased the binding affinity of autoantibodies. Additionally, the exclusive recognition by patients of only one of the epitopes within this region suggests a genetic restriction of the autoantibody response.