CD4CD25 Tregs play a central role in the maintenance of peripheral self tolerance by keeping autoreactive T cells in check. Whereas the thymic origin of CD4CD25 Tregs, as a distinct lineage, has been inferred, understanding of their developmental pathways has remained elusive. In both mice and humans, peripheral CD4CD25 Treg populations have been described as composed of antigen-experienced T cells that fail to significantly proliferate following TCR stimulation but suppress proliferation and effector functions of CD25 T cells. Here we show that analysis of CD25 expression in human circulating CD4 T lymphocytes with respect to their in vivo differentiation stages identifies a distinct subset of CD25CCR7CD62LCTLA-4FOXP3 cells contained in the CD45RA/RO naive fraction. The subset, which we have named natural naive Tregs (NnTregs), is prominent in young adults and decreases with age together with the total naive CD4 population. NnTregs are anergic following stimulation in the absence of IL-2 and exert ex vivo cell-cell contact-mediated suppressor functions. In addition, they proliferate in response to stimulation with autologous APCs, which indicates a high enrichment in T cells bearing self-reactive TCRs. The definition of this subset has important implications for the analysis of human naturally occurring Tregs and for their targeting in therapeutic immune interventions.