NOD.H-2h4 mice are genetically predisposed to thyroid autoimmunity and spontaneously develop thyroglobulin autoantibodies (TgAb) and thyroiditis. Iodide administration enhances TgAb levels and the incidence and severity of thyroiditis. Using these mice, we investigated the interactions between TSH receptor (TSHR) antibodies induced by vaccination and spontaneous or iodide-enhanced thyroid autoimmunity (thyroiditis and TgAb). Mice were immunized with adenovirus expressing the TSHR A-subunit (or control adenovirus). Thyroid antibodies, histology, and serum thyroxine levels were compared in animals on a regular diet or on a high-iodide diet (0.05% NaI-supplemented water). Thyroiditis severity and TgAb levels were enhanced by iodide administration and were independent of the type of adenovirus used for immunization. In contrast, TSHR antibodies, measured by TSH-binding inhibition, thyroid-stimulating activity, and TSH-blocking activity, were induced in the majority of animals immunized with TSHR (but not control) adenovirus and were unaffected by dietary iodide. The NOD.2h4 strain of mice was less susceptible than BALB/c or BALB/k mice to TSHR adenovirus-induced hyperthyroidism. Nevertheless, hyperthyroidism developed in approximately one third of TSHR adenovirus-injected NOD.2h4 mice. This hyperthyroidism was suppressed by a high-iodide diet, probably by a nonimmune mechanism. The fact that inducing an immune response to the TSHR had no effect on thyroiditis raises the possibility that the TSHR may not be the target involved in the variable thyroiditis component in some humans with Graves' disease.