We showed previously that cecal bacterial Ag (CBA)-specific CD4+ T cells induce colitis when transferred into SCID mice. The purpose of this study was to generate and characterize CBA-specific regulatory T cells in C3H/HeJBir (Bir) mice. CD4+ T cells were stimulated with CBA-pulsed APC in the presence of IL-10 every 10–14 days. After four or more cycles, these T cells produced high levels of IL-10, low levels of IL-4 and IFN-γ, and no IL-2, consistent with the phenotype of T regulatory-1 (Tr1) cells. Bir Tr1 cells proliferated poorly, but their proliferation was dependent on CD28-B7 interactions and was MHC class II-restricted. Transfer of Bir Tr1 cells into SCID mice did not result in colitis, and cotransfer of Bir Tr1 T cells with pathogenic Bir CD4+ Th1 cells prevented colitis. Bir Tr1 cells inhibited proliferation and IFN-γ production of a CBA-specific Th1 cell line in vitro. Such inhibition was partly due to IL-10 and TGFβ1, but cognate interactions with either APCs or Th1 cells were also involved. Normal intestinal lamina propria CD4+ T cells had Tr1-like activity when stimulated with CBA-pulsed APCs. We conclude that CD4+ T cells with the properties of Tr1 cells are present in the intestinal lamina propria and hypothesize that these cells maintain intestinal immune homeostasis to the enteric flora.