The forkhead/winged helix transcription factor (Foxp3) is expressed as two different isoforms in humans: the full-length isoform (Foxp3FL) and an alternative-splicing product lacking the exon 2 (Foxp3DeltaE2). We here studied the cellular distribution of Foxp3 isoforms by quantitative PCR and evaluated the functional outcome of retroviral transduction of Foxp3FL and Foxp3DeltaE2 genes into CD4(+)CD25(-) cells. In PBMC, both isoforms were preferentially expressed in CD4(+)CD25(hi) cells. In single-cell-sorted and expanded Treg, both Foxp3 isoforms were expressed simultaneously but without a fixed ratio. Forced expression of Foxp3FL or Foxp3DeltaE2 genes in CD4(+)CD25(-) T cells induced bona fide Treg that not only displayed Treg phenotype but also were anergic and mediated significant suppressive activity against CD3-activated CD4(+)CD25(-) cells. GFP(-) nontransduced cells or cells transduced with an empty vector showed no Treg phenotype, anergy or suppressive activities. In conclusion, our results reveal that both Foxp3 isoforms possess similar capacities to induce Treg; however, unnaturally high expression levels are required to convey Treg functions to CD4(+)CD25(-) cells. As both Foxp3 isoforms appear to be expressed in an independent fashion, studies aiming at quantification of Treg in peripheral blood or in tissue samples can benefit from determination of total Foxp3 levels rather than one of the isoforms.