The transcriptional regulator FOXP3 is an important determinant of regulatory T (Treg) cell development and function and is frequently used to quantitate Treg cells. However, FOXP3 is also expressed in recently activated conventional human T cells. Here, we investigated the FOXP3 expression patterns in Treg and activated T cells at a cellular level. Upon activation, human CD4+CD25− T cells expressed FOXP3 mainly in the cytoplasm, in sharp contrast to human CD4+CD25+ Treg cells, where we found FOXP3 to be predominantly expressed in the nucleus. A GFP-FOXP3-fusion protein shuttled from the nucleus to the cytoplasm in transfected primary human T cells. We identified two novel leucine-rich nuclear export signals in FOXP3. Site-directed mutagenesis of both sequences completely abolished nuclear export of FOXP3 in human T cells. Both export sequences localized to exons affected by alternative splicing. The three isoforms FOXP3Δ2, FOXP3Δ7, and FOXP3Δ2Δ7 localized preferentially to the nucleus. Additionally, forced expression of nucleus-directed FOXP3 induced a Treg-cell-associated gene expression pattern and induced regulatory capacity. These findings should aid in the interpretation of future studies utilizing FOXP3 expression as a Treg-cell marker and shed some light on the molecular mechanisms controlling subcellular FOXP3 localization in human T cells.