Autoimmune thyroiditis (AT) is characterized by a continuous inflammatory self-destructive process that eventually leads to chronic progressive dysfunction of the thyroid. In a previously established experimental AT model, C57bl/6 mice immunized with recombinant mouse thyroid peroxidase (TPO) (rmTPO) developed lymphocytic thyroiditis and anti-TPO antibody but not chronic hypothyroidism. To determine the immunodominant epitope(s) of TPO, T cell proliferation assays were performed in which rmTPO-primed lymph nodes cells were reacted with recombinant mTPO fragments or short overlapping synthetic TPO peptides. Within residue 405-849, peptide 540-559 gave the maximum proliferation response with a stimulation index more than 12. Mice immunized with peptide 540-559 developed antibody against rmTPO and native mouse TPO protein, lymphocytic thyroiditis, and hypothyroidism. In conclusion, this study demonstrated that TPO is the autoantigen for the development of lymphocyte thyroiditis and thyroid dysfunction, and peptide 540-559 is the immunodominant T cell epitope of TPO. Identification of T cell epitopes of TPO may enable the development of immunotherapy to prevent chronic hypothyroidism in AT.