In previous studies, we have shown that murine CD4+ CD25+ regulatory T cells produce high levels of TGF-β1 in a cell surface and/or secreted form, and blockade of such TGF-β1 by anti-TGF-β curtails the ability of these cells to suppress CD25− T cell proliferation and B cell Ig production in in vitro suppressor assays. In further support for the role of TGF-β1 in suppression by CD4+ CD25+ T cells, we show in this study that another TGF-β1-blocking molecule, recombinant latency-associated peptide of TGF-β1 (rLAP), also reverses suppression by mouse CD4+ CD25+ T cells as well as their human counterparts, CD4+ CD25 high T cells. In addition, we show that CD25− T cells exposed to CD4+ CD25+ T cells in vitro manifest activation of Smad-2 and induction of CD103, the latter a TGF-β-inducible surface integrin. In further studies, we show that while CD4+ CD25+ T cells from TGF-β1-deficient mice can suppress CD25− T cell proliferation in vitro, these cells do not protect recipient mice from colitis in the SCID transfer model in vivo, and, in addition, CD4+ LAP+, but not CD4+ LAP− T cells from normal mice protect recipient mice from colitis in this model. Together, these studies demonstrate that TGF-β1 produced by CD4+ CD25+ T cells is involved in the suppressor activity of these cells, particularly in their ability to regulate intestinal inflammation.