Human anti-thyroperoxidase (TPO) autoantibodies (aAbs) are a major hallmark of autoimmune thyroid diseases. Their epitopes are discontinuous and mainly restricted to an immunodominant region (IDR) consisting of two overlapping regions (IDR/A and B). To shed light on the relationship between these regions, we first performed competitive studies using all available reference anti-TPO antibodies. Interestingly, we showed that human IDR/A- and B-specific anti-TPO aAbs recognized essentially the same regions on the TPO molecule. However, our data also indicated that IDR/A-specific human aAbs strongly recognized the region containing residues 599-617, whereas the IDR/B-specific aAbs bind to several regions as well as region 599-617. Next, we scanned this key region to identify the residues involved in the immunodominant autoepitope. Using peptide spot technology together with competitive ELISA experiments, we demonstrated that residues (604)ETP-DL(609) play a major role in the anti-peptide P14 epitope and that IDR/A-specific human anti-TPO aAbs, either expressed as recombinant Fab or obtained from Graves' disease patients, specifically recognize the sequences (597)FCGLPRLE(604) and (611)TAIASRSV(618). All together our data emphasize that both the IDRs involve the same surface area on human TPO, but the differential usage of one or the other regions leads to different inhibition patterns in competitive experiments. In conclusion, our data help to resolve the long-sought issue on the molecular immunology of the two IDRs on TPO and provide new clues to design efficient peptides that may be part of a combinatorial treatment aiming at delaying development of autoimmune thyroiditis when used prophylactically.