Oral administration of myelin basic protein (MBP) is an effective means of suppressing experimental autoimmune encephalomyelitis (EAE). In the Lewis rat model, we have previously shown that this effect is mediated by active suppression as T lymphocytes from animals orally tolerized to MBP suppress in vitro immune responses and in vivo adoptively transfer disease protection to naive recipients. This effect is mediated by the cytokine TGF-beta which is secreted by T cells from orally tolerized animals after being triggered by the oral tolerogen. In the present study we investigated Peyer's patches in SJL mice following orally administered MBP. Peyer's patches are one of the major lymphoid structures of gut-associated lymphoid tissue, and a site thought to play an important role in the induction of oral tolerance. Twenty-four hours after one feeding of 1 mg of MBP, there were no proliferative responses to MBP in Peyer's patches. However, when Peyer's patches from MBP-fed animals were stimulated with IL-2 in the presence of MBP, reduced proliferation to IL-2 was observed, and this inhibition was reversed with anti-TGF-beta antibody. Suppression of IL-2-induced proliferation by MBP was not observed in unfed animals or if Peyer's patches from MBP-fed animals were stimulated with a control antigen (ovalbumin). Stimulation of Peyer's patches T cells from MBP-fed animals with MBP resulted in secretion of TGF-beta in a dose-related fashion with less TGF-beta secretion at higher doses. Furthermore, cells from Peyer's patches of animals fed MBP adoptively transferred protection to actively induced EAE. Thus, MBP-specific TGF-beta-secreting regulatory cells recovered from Peyer's patches after a single oral administration of MBP are not evident as measured by proliferation, but are capable of suppressing in vitro and in vivo cell-mediated immune responses. Peyer's patches appear to be an important site for the induction of cells which mediate the active suppression component of oral tolerance.