A CD4⁺ T cell subpopulation defined by the expression levels of a particular cell surface molecule (e.g. CD5, CD45RB, CD25, CD62L or CD38) bears an autoimmune-preventive activity in various animal models. Here we show that the expression of CD25 is highly specific, when compared with other molecules, in delineating the autoimmune-preventive immunoregulatory CD4⁺ T cell population. Furthermore, although CD25 is an activation marker for T cells, the following findings indicate that immunoregulatory CD25⁺CD4⁺ T cells are functionally distinct from activated or anergy-induced T cells derived from CD25^–CD4⁺ T cells. First, the former are autoimmune-preventive in vivo, naturally unresponsive (anergic) to TCR stimulation in vitro and, upon TCR stimulation, able to suppress the activation/proliferation of other T cells, whereas the latter scarcely exhibit the in vivo autoimmune-preventive activity or the in vitro suppressive activity. Second, such activated or anergy-induced CD25^– spleen cells produce various autoimmune diseases when transferred to syngeneic athymic nude mice, whereas similarly treated normal spleen cells, which include CD25⁺CD4⁺ T cells, do not. Third, upon polyclonal T cell stimulation, CD25⁺CD4⁺ T cells express CD25 at higher levels and more persistently than CD25^–CD4⁺ T cell-derived activated T cells; moreover, when the stimulation is ceased, the former revert to the original levels of CD25 expression, whereas the latter lose the expression. These results collectively indicate that naturally anergic and suppressive CD25⁺CD4⁺ T cells present in normal naive mice are functionally and phenotypically stable, distinct from other T cells, and play a key role in maintaining immunologic self-tolerance.