Murine CD4+ CD25+ regulatory cells have been reported to express latency-associated peptide (LAP) and TGF-β on the surface after activation, and exert regulatory function by the membrane-bound TGF-β in vitro. We have now found that a small population of CD4+ T cells, both CD25+ and CD25−, can be stained with a goat anti-LAP polyclonal Ab without being stimulated. Virtually all these LAP+ cells are also positive for thrombospondin, which has the ability to convert latent TGF-β to the active form. In the CD4+ CD45RB high-induced colitis model of SCID mice, regulatory activity was exhibited not only by CD25+ LAP+ and CD25+ LAP− cells, but also by CD25− LAP+ cells. CD4+ CD25− LAP+ T cells were part of the CD45RB low cell fraction. CD4+ CD25− LAP− CD45RB low cells had minimal, if any, regulatory activity in the colitis model. The regulatory function of CD25− LAP+ cells was abrogated in vivo by anti-TGF-β mAb. These results identify a new TGF-β-dependent regulatory CD4+ T cell phenotype that is CD25− and LAP+.