Autoantibodies (aAbs) to thyroid peroxidase (TPO), the hallmark of autoimmune thyroid disease (AITD), recognize conformational epitopes restricted to an immunodominant region (IDR), divided into two overlapping domains A and B. Despite numerous efforts aimed at localizing the IDR and identifying aAb-interacting residues on TPO, only two critical amino acids, Lys 713 and Tyr 772 , have been characterized. Precise and complete delineation of the other residues involved in the IDR remains to be defined. By using a recombinant anti-TPO aAb T13, we demonstrated that four regions on TPO are part of the IDR/B; one of them, located between amino acids 713 and 720, is particularly important for the binding of sera from patients suffering from AITD. To precisely define critical residues implicated in the binding of aAb to human TPO, we used directed mutagenesis and expressed the mutants in stably transfected CHO cells. Then we assessed the kinetic parameters involved in the interactions between anti-TPO aAbs and mutants by real-time analysis. We identified (i) the minimal epitope 713-717 recognized by mAb 47 (a reference antibody) and (ii) the amino acids used as contact points for two IDR-specific human monoclonal aAbs TR1. Thyroid peroxidase (TPO) 1 is an essential membrane-bound enzyme involved in the biosynthesis of iodinated thyroid hormones. As well as thyroglobulin and the thyrotropin receptor, TPO is a major thyroid autoantigen (aAg) targeted by the immune system during autoimmune thyroid diseases (AITD). Human TPO (hTPO) is a 933 amino acid molecule and possesses a highly convoluted three-dimensional structure formed by three distinct modules, respectively, homologous with the myeloperoxidase (MPO), the complement control protein (CCP), and the epidermal growth factor (EGF) (1). This architectural complexity is a strong obstacle for producing high resolution crystals (2, 3), explaining why the only available three-dimensional model has been generated by computer approaches (1). Autoantibodies (aAbs) against hTPO, strongly expressed in patients' sera, are sensitive and specific diagnostic markers for AITD and may play a role in the process leading to thyroid cellular dysfunction and destruction (4-8). More importantly, anti-TPO aAbs have been found to be involved in antigen presentation to autoaggressive T cells (9) and may consequently enhance AITD as previously shown for antithyroglobulin aAbs (10). Therefore, a precise delineation of the amino acids constituting the epitopes recognized by human aAbs on TPO would be very helpful for understanding how this aAg is recognized during the onset of AITD, as well as for developing specific immune interventions to prevent or block such pathologies.A high proportion of anti-TPO aAbs from patients' sera recognizes conformational and discontinuous epitopes, restricted to an immunodominant region (IDR) composed of two overlapping domains called A and B (11-13). Several strategies have been used to localize these domains and, in particular, the critical amino acids involve...