Thyroid peroxidase (TPO) is a major autoantigen in thyroid autoimmune disease where pathogenic autoantibodies recognise conformational epitopes restricted to two overlapping immunodominant regions (IDR) termed IDR-A and -B. Based upon our structural model of TPO, we report on the localisation of the IDRs to specific amino acids important for autoantibody binding. Using a panel of recombinant human Fabs (rhFabs) from autoimmune patients, specific for the IDR-A or -B epitopes, in combination with eukaryotic expression of 14 single amino acid mutants of TPO, we identify R225 and K627 as key components of IDR-A and -B, respectively. Moreover, each mutant specifically led to the loss of binding of three different IDR-A- or -B-specific rhFabs, without affecting the binding of autoantibodies to the other determinant. Further supportive evidence for the role of amino acids R225 and K627 was obtained with murine monoclonal antibodies that first defined the IDRs. The identification of amino acids R225 and K627 as key residues for the IDR epitopes on TPO will advance our understanding of the molecular basis of autoreactivity and facilitate the design of novel therapeutic agents.