This study shows that the normal thymus produces immunoregulatory CD25+ 4+ 8− thymocytes capable of controlling self-reactive T cells. Transfer of thymocyte suspensions depleted of CD25+ 4+ 8− thymocytes, which constitute∼ 5% of steroid-resistant mature CD4+ 8− thymocytes in normal naive mice, produces various autoimmune diseases in syngeneic athymic nude mice. These CD25+ 4+ 8− thymocytes are nonproliferative (anergic) to TCR stimulation in vitro, but potently suppress the proliferation of other CD4+ 8− or CD4− 8+ thymocytes; breakage of their anergic state in vitro by high doses of IL-2 or anti-CD28 Ab simultaneously abrogates their suppressive activity; and transfer of such suppression-abrogated thymocyte suspensions produces autoimmune disease in nude mice. These immunoregulatory CD25+ 4+ 8− thymocytes/T cells are functionally distinct from activated CD25+ 4+ T cells derived from CD25− 4+ thymocytes/T cells in that the latter scarcely exhibits suppressive activity in vitro, although both CD25+ 4+ populations express a similar profile of cell surface markers. Furthermore, the CD25+ 4+ 8− thymocytes appear to acquire their anergic and suppressive property through the thymic selection process, since TCR transgenic mice develop similar anergic/suppressive CD25+ 4+ 8− thymocytes and CD25+ 4+ T cells that predominantly express TCRs utilizing endogenous α-chains, but RAG-2-deficient TCR transgenic mice do not. These results taken together indicate that anergic/suppressive CD25+ 4+ 8− thymocytes and peripheral T cells in normal naive mice may constitute a common T cell lineage functionally and developmentally distinct from other T cells, and that production of this unique immunoregulatory T cell population can be another key function of the thymus in maintaining immunologic self-tolerance.