Previous studies have shown that autoimmune thyroiditis can be induced in normal laboratory rats after thymectomy and split dose γ-irradiation. Development of disease can be prevented by reconstitution of PVG rats shortly after their final irradiation with either peripheral CD4⁺CD45RC⁻ T cells or CD4⁺CD8⁻ thymocytes from syngeneic donors. Although the activity of both populations is known to depend on the activities of endogenously produced interleukin 4 and transforming growth factor β, implying a common mechanism, the issue of antigen specificity of the cells involved has not yet been addressed. In this study, we show that the regulatory T cells that prevent autoimmune thyroiditis are generated in vivo only when the relevant autoantigen is also present. Peripheral CD4⁺ T cells, from rats whose thyroids were ablated in utero by treatment with ¹³¹I, were unable to prevent disease development upon adoptive transfer into thymectomized and irradiated recipients. This regulatory deficit is specific for thyroid autoimmunity, since CD4⁺ T cells from ¹³¹I-treated PVG.RT1^u rats were as effective as those from normal donors at preventing diabetes in thymectomized and irradiated PVG.RT1^u rats. Significantly, in contrast to the peripheral CD4⁺ T cells, CD4⁺CD8⁻ thymocytes from ¹³¹I-treated PVG donors were still able to prevent thyroiditis upon adoptive transfer. Taken together, these data indicate that it is the peripheral autoantigen itself that stimulates the generation of the appropriate regulatory cells from thymic emigrant precursors.