Oral administration of antigen induces a systemic hyporesponsiveness termed oral tolerance. High doses of oral antigen lead to deletion or anergy of T-cells whereas low doses induce regulatory T-cells that secrete Th2 cytokines (IL-4/IL-10) and TGF-beta. The initiating events associated with oral tolerance have not been well characterized. We investigated the induction phase of oral tolerance by adoptively transferring ovalbulumin (OVA) p (323-339) TcR specific transgenic (Tg+) T-cells into BALB/c recipients that were then fed either a high (5 mg x 5) or a low (0.1 mg x 5) dose of OVA 323-329 peptide. The frequency of Tg+ T-cells in lymphoid tissues was determined by flow cytometry using an anti-clonotypic monoclonal antibody. In high-dose-fed animals, Tg+ cells increased six- to eightfold in Peyer's patches after one feeding and then progressively decreased to 44% of those in the control by Day 20. In contrast, a biphasic-type response was observed in lymph node and spleen where Tg+ cells decreased after the first feeding, returned to the control level, and then decreased to 36-63% of the control level by Day 20. In low-dose-fed animals, changes in Tg+ T cells were only observed in Peyer's patches after five feedings, where cells increased approximately twofold. T-cell activation as measured by proliferation and IFN-gamma secretion occurred in both low- and high-dose-fed animals after only one feeding and then declined whereas secretion of Th2 cytokines and TGF-beta remained high even 10 days after the last feeding in low-dose-fed animals. Immunization with OVA/CFA demonstrated peripheral tolerance as measured by decreased proliferation and IFN-gamma secretion and was associated with increased production of TGF-beta and IL-10. These results suggest that the inductive phase of oral tolerance is characterized by an activation of antigen-specific T-cells that involves the initial secretion of IFN-gamma followed by prolonged secretion of Th2 cytokines and TGF-beta.