CD4⁺ T cells play a vital role in mediating the tolerance induced at mucosal sites following exposure to non‐pathogenic stimuli, and further understanding of the precise mechanisms bywhich these cells prevent aberrant responses is required. We have developed a model using transfer of DO11.10 TCR‐transgenic bone marrow into irradiated recipients in which it has been possible to track antigen‐specific CD4⁺ cells in mesenteric lymph nodes (mLN), Peyer's patches (PP) and lamina propria following primary exposure to antigen. Using this model we have demonstrated initial activation in all three gut‐associated lymphoid tissue compartments characterized by increases in the frequency of transgenic cells expressing CD69 and CD25. These cells subsequently enter astate of hyporesponsiveness both locally in the mLN and PP and in the periphery following feeding and challenge. Investigating the role of CTLA‐4 either using anti‐CTLA‐4 mAb or by generating chimeras using DO11.10×CTLA‐4^–/– mice as donors we have clearly shown that antigen‐specific cells require the expression of this regulatory molecule for oral tolerance. In contrast, oral tolerance was intact in chimeras generated using DO11.10×IL‐10^–/– cells, indicating that secretion of this cytokine by antigen‐specific cells is not required.