Orally applied antigens generate systemic unresponsiveness by induction of anergy and deletion of specific T cells at high antigen doses, and induction of regulatory T cells at low doses of antigen. These different immune reactions have been attributed to different types of antigen-presenting cells (APC) and/or different secondary lymphoid organs participating in the induction of the immune response. We used high-sensitivity immunofluorescence to directly identify for the first time the cells presenting orally applied antigen in vivo. At low peptide doses (<1 mg) peptide presentation was exclusively detected on dendritic cells (DC) of the Peyer's patches (PP) and mesenteric lymph nodes (mLN). At high doses (>1 mg) peptides were presented systemically and by all types of APC but presentation was still maximal on DC of the PP (up to 65%). Nevertheless, at limiting antigen doses T cell activation in the gut-associated lymphoid tissue occurs preferentially in the mLN but not in PP. PP-deficient mice have the same frequencies of peptide-presenting cells in mLN, peripheral lymph nodes and spleen and activation of naive T cells in vivo is not affected. Therefore, PP are not critical for antigen presentation as well as for T cell activation in response to orally applied soluble antigens.