Despite thymic deletion of cells with specificity for self-antigens, autoreactive T cells are readily detectable in the normal T-cell repertoire. In recent years, a population of CD4⁺ T cells that constitutively express the interleukin-2 receptor-α chain, CD25, has been shown to play a pivotal role in the maintenance of self-tolerance in rodent models. This study investigated whether such a regulatory population exists in humans. A population of CD4⁺CD25⁺ T cells, taken from the peripheral blood of healthy individuals and phenotypically distinct from recently activated CD4⁺ T cells, was characterized. These cells were hyporesponsive to conventional T-cell stimuli and capable of suppressing the responses of CD4⁺CD25⁻ T cells in vitro. Addition of exogenous interleukin-2 abrogated the hyporesponsiveness and suppressive effects of CD4⁺CD25⁺ cells. Suppression required cell-to-cell contact but did not appear to be via the inhibition of antigen-presenting cells. In addition, there were marked changes in the expression of Notch pathway molecules and their downstream signaling products at the transcriptional level, specifically in CD4⁺CD25⁺ cells, suggesting that this family of molecules plays a role in the regulatory function of CD4⁺CD25⁺ cells. Cells with similar phenotype and function were detected in umbilical venous blood from healthy newborn infants. These results suggest that CD4⁺CD25⁺ cells represent a population of regulatory T cells that arise during fetal life. Comparison with rodent CD4⁺CD25⁺ cells suggests that this population may play a key role in the prevention of autoimmune diseases in humans.