Thyroid peroxidase (TPO) autoantibody epitopes are largely restricted to an immunodominant region (IDR) on the extracellular region of the native molecule. Localization of the IDR has been a longstanding and difficult goal. The TPO extracellular region comprises a large myeloperoxidase-like domain, linked to the plasma membrane by two smaller domains with homology to complement control protein (CCP) and epidermal growth factor (EGF), respectively. Recent studies have focused on the CCP-and EGF-like domains as the putative location of the TPO autoantibody IDR. To address this issue, we attempted to express on the surface of transfected cells native TPO in which the CCP-and EGF-like domains were deleted, either together or individually. We used a quartet of human monoclonal autoantibodies that define the TPO IDR, as well as polyclonal TPO autoantibodies in patients' sera, to detect these mutated TPO molecules by flow cytometry. The combined CCP/EGF-like domain deletion did not produce a signal with TPO autoantibodies but did not traffic to the cell surface. In contrast, both monoclonal and polyclonal autoantibodies recognized TPO with the juxtamembrane EGF-like domain deleted equally as well as the wild-type TPO on the cell surface. TPO with the CCP-like domain deleted expressed normally on the cell surface, as determined using the polyclonal mouse antiserum. Nevertheless, this modified TPO molecule was recognized very poorly by both the human monoclonal autoantibodies and the polyclonal autoantibodies in patients' sera. In conclusion, we have clearly excluded the juxtamembrane EGF-like domain as being part of the IDR. In contrast, a component of the CCP-like domain does contribute to the IDR. These data, together with findings from other studies, localize the TPO autoantibody IDR to the junction of the CCP-like domain and the much larger myeloperoxidase-like domain on TPO.Thyroid peroxidase (TPO), 1 a heme-containing glycoprotein on the thyrocyte apical membrane that plays a key role in thyroid hormone biosynthesis, is also the dominant autoantigen in human autoimmune thyroiditis (1-3). A remarkable feature of polyclonal TPO autoantibodies in the sera of all patients is that their epitopes are largely directed to a restricted area on the native antigen, termed the immunodominant region (IDR)(reviewed in Ref. 4). This epitopic profile contrasts with antibodies generated in mice by immunization with purified TPO together with adjuvant, which have a wide range of epitopes on both native and denatured TPO (5). Even within the IDR, TPO autoantibodies in an individual patient maintain the same epitopic fingerprint over many years (lack of epitope spreading) (6), a phenomenon that appears to have a genetic basis (7).The role of TPO autoantibodies in the pathogenesis of disease is debated. Some evidence supports their involvement in thyrocyte damage by antibody-mediated cellular cytotoxicity (8, 9). Perhaps more important is the influence of antibodies complexed to antigen in modulating antigen processing with enhancemen...