CD4+ CD25+ regulatory T cells (Treg) are potent suppressors, and play important roles in autoimmunity and transplantation. Recent reports suggest that CD4+ CD25+ Treg are not a homogeneous cell population, but the differences in phenotype, function, and mechanisms among different subsets are unknown. Here, we demonstrate CD4+ CD25+ Treg cells can be divided into subsets according to cell-surface expression of CD62L. While both subsets express foxp3 and are anergic, the CD62L+ population is more potent on a per cell basis, and proliferates and maintains suppressive function far better than the CD62L- population and unseparated CD4+ CD25+ Treg. The CD62L+ population preferentially migrates to CCL19, MCP-1 and FTY720. Both CD62L+ and CD62L- subsets prevent the development of autoimmune gastritis and colitis induced by CD4+ CD25-CD45RBhigh cells in severe combined immunodeficiency (SCID) mice. Overall, these results suggest CD4+ CD25+ Treg are not a homogenous cell population, but can be divided into at least two subsets according to CD62L expression. The CD62L+ subset is a more potent suppressor than the CD62L- population or unfractionated CD4+ CD25+ Treg cells, can be expanded far more easily in culture, and is more responsive to chemokine-driven migration to secondary lymphoid organs. These properties may have significant implications for the clinical manipulation of the CD4+ CD25+ CD62L+ cells.