Graves' hyperthyroidism can be efficiently induced in susceptible mouse strains by repeated immunization with recombinant adenovirus coding the TSH receptor (TSHR). This study was designed to evaluate the role(s) played by naturally occurring CD4⁺CD25⁺ regulatory T cells in the development of Graves' hyperthyroidism in resistant C57BL/6 and susceptible BALB/c mice. Depletion of CD4⁺CD25⁺ T cells rendered some C57BL/6 mice susceptible to induction of hyperthyroidism. Thus, hyperthyroidism developed in 30% CD4⁺CD25⁺ T cell-depleted C57BL/6 mice immunized with adenovirus expressing the TSHR A-subunit (AdTSHR289) vs. 0% in those immunized with AdTSHR289 alone. This immunological manipulation also enhanced disease severity in susceptible BALB/c mice, as reflected by a significant increase in mean thyroxine levels by CD4⁺CD25⁺ T cell-depletion. The immuno-enhancing effect of CD4⁺CD25⁺ T cell-depletion appears to be attributed to an increase in thyroid stimulating antibody production and/or a decrease in thyroid blocking antibody synthesis, but not immune deviation to either Th1 or Th2. Interestingly, unlike BALB/c mice, some hyperthyroid C57BL/6 mice showed some intrathyroidal lymphocytic infiltration with follicular destruction. These results indicate that CD4⁺CD25⁺ T cells play a role in disease susceptibility and severity in adenovirus-TSHR induced Graves' hyperthyroidism. Overall the imbalance between effector and regulatory T cells appears to be crucial in the pathogenesis of Graves' disease.