Thyroid peroxidase (TPO) is a major autoantigen of thyroid autoimmune disease, and the autoantibodies that are produced recognize two immunodominant regions (IDR) of the molecule, termed IDR-A and -B. Based upon our structural model of the TPO ectodomain, we recently identified R225 and K627 as key residues in IDR-A and -B, respectively. We report here on rational mutagenic investigations to identify additional residues surrounding R225 and K627 that affect the binding of recombinant human Fabs (rhFabs) specific for each IDR. Two residues R646 and D707 were identified from the model as promising surface-exposed amino acids adjacent to R225. Similarly, residues E604, D620, D624, and D630 were identified in the vicinity of K627. These residues were substituted in different combinations of single, double, and multiple mutations, and stably expressed in Chinese hamster ovary cells. By fluorescence-activated cell sorting and capture ELISA, we found that R225A, R646A, and D707N specifically led to the loss of binding of IDR-A rhFabs, whereas E604A, D620R, K627G, and D630N specifically abrogated the binding of IDR-B rhFabs. Further supportive evidence of the importance of these residues for the IDR epitopes was obtained with patients' sera. We conclude that R646 and D707 together with R225 constitute a functional epitope within IDR-A, and that residues E604, D620, and D630, together with K627, constitute a functional epitope within IDR-B. This identification of key residues within the autoreactive epitopes will help in understanding the structural basis for the breakdown of immune tolerance to TPO in thyroid autoimmune disease.