Naive lymphocytes systemically recirculate for immunosurveillance inspecting foreign antigens and pathogens in the body. Trafficking behavior such as the migration pathway and transit time within the gastrointestinal tract, however, remains to be elucidated. Rat thoracic duct lymphocytes (TDLs) were transferred to a congeneic host that had undergone mesenteric lymphadenectomy. The migration pathway was investigated using newly developed four-color immunohistochemistry and immunofluorescence. Donor TDLs showed rapid transition in gut tissues from which they emerged in mesenteric lymph around 4 h after intravenous injection. Immunohistochemistry showed that donor TDLs predominantly transmigrated across high endothelial venules (HEVs) at the interfollicular area of the Peyer's patches (PPs), then exited into the LYVE-1+ efferent lymphatics, that were close to the venules. The rapid recirculation depended largely on the local expression of unsulfated sialyl-Lewis X on these venules where putative dendritic cells (DCs) were associated underneath. Recruited naive T cells briefly made contact with resident DCs before exiting to the lymphatics in the steady state. In some transplant settings, however, the T cells retained contact with DCs and were sensitized and differentiated into activated T cells. In conclusion, we directly demonstrated that lymphocyte recirculation within the gut is a very rapid process. The interfollicular area of PPs functions as a strategically central site for rapid immunosurveillance where HEVs, efferent lymphatics and resident DCs converge. PPs can, however, generate alloreactive T cells, leading to exacerbation of graft-versus-host disease or gut allograft rejection.