The transcription factor FOXP3 plays a key role in CD4⁺CD25⁺ regulatory T cell function and represents a specific marker for these cells. Despite its strong association with regulatory T cell function, in humans little is known about the frequency of CD4⁺CD25⁺ cells that express FOXP3 protein nor the distribution of these cells in vivo. Here we report the characterization of seven anti‐FOXP3 monoclonal antibodies enabling the detection of endogenous human FOXP3 protein by flow cytometry and immunohistochemistry. Flow‐cytometric analysis showed that FOXP3 was expressed by the majority of CD4⁺CD25^high T cells in peripheral blood. By contrast, less than half of the CD4⁺CD25^int population were FOXP3⁺, providing an explanation for observations in human T cells that regulatory activity is enriched within the CD4⁺CD25^high pool. Although FOXP3 expression was primarily restricted to CD4⁺CD25⁺ cells, it was induced following activation of both CD4⁺ and CD8⁺ T cell clones. These findings indicate that the frequency of FOXP3⁺ cells correlates with the level of expression of CD25 in naturally arising regulatory T cells and that FOXP3 protein is expressed by some activated CD4⁺ and CD8⁺ T cell clones. These reagents represent valuable research tools to further investigate FOXP3 function and are applicable for routine clinical use.

See accompanying Commentary http://dx.doi.org/10.1002/eji.200526303