CD25+CD4+ regulatory T cells (Treg) play pivotal roles in the host response to tumors. However, their exact location in vivo is largely unknown. The forkhead/winged helix transcription factor, Foxp3, is specifically expressed in naturally occurring Treg (nTreg) and programs their development and function. In this study, we produced a rabbit polyclonal antibody (pAb), which can detect mouse Foxp3 protein in situ. Results using this pAb revealed that Foxp3+CD4+ nTreg cells occur in direct contact with CD11c+ dendritic cells (DCs), and Foxp3-CD4+ and CD8+T lymphocytes in the T cell regions of lymphoid tissues from normal and tumor-bearing mice. The numbers of Foxp3+CD4+ nTreg cells are significantly increased in draining, but not nondraining, lymph nodes (LNs) and spleen (SPL) of tumor-bearing mice. Furthermore, a small number of nTreg could be also found at the tumor site. These observations support the notion that the numbers of Foxp3+CD4+ nTreg are increased by tumors and may contribute to the immunosuppression observed in tumor-bearing hosts at secondary lymphoid organs and also possibly at the tumor site.