Regulatory T cells (TREGs) constitutively expressing CD4, CD25, and the transcription factor Foxp3 can prevent a wide range of experimental and spontaneous autoimmune diseases in mice. In humans, CD4⁺CD25^bright T cells, predominantly within the CD45RO⁺ activated/memory subset in adults and the CD45RA⁺ naive T-cell subset in infants, are considered to be the equivalent subset. Using novel combinations of monoclonal antibodies (mAbs), we examined expression of CD25 in human infant thymus, cord blood, adult peripheral blood, lymph node, and spleen. In addition to the CD4⁺CD25^bright T cells, subfractionation on the basis of CD45 splice variants indicated that all samples contained a second distinct population of cells expressing a slightly lower level of CD25. In adult peripheral blood, this population expressed a naive CD45RA⁺ phenotype. The corresponding population in lymph node, spleen, and cord blood showed some evidence of activation, and expressed markers characteristic of TREGs, such as cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). Sorted CD4⁺CD25⁺CD45RA⁺ T cells from both cord and adult blood expressed very high levels of mRNA for Foxp3 and manifested equivalent suppressive activity in vitro, indicating that they are bone fide members of the regulatory T-cell lineage. Targeting naive TREGs in adults may offer new means of preventing and treating autoimmune disease.