CD4⁺CD25⁺ T cells in mice and rats are capable of transferring protection against organ‐specific autoimmune disease and colitis and suppressing the proliferation of other T cells after polyclonal stimulation in vitro. Here we describe the existence in humans of CD4⁺CD25⁺ T cells with the same in vitro characteristics. CD4⁺CD8^–CD25⁺ T cells are present in both the thymus and peripheral blood of humans (∼ 10 % of CD4⁺CD8^– T cells), proliferate poorly in response to mitogenic stimulation and suppress the proliferation of CD4⁺CD25^– cells in co‐culture. This suppression requires cell contact and can be overcome by the addition of exogenous IL‐2. CD4⁺CD25⁺ cells from thymus and blood were poor producers of IL‐2 and IFN‐γ, and suppressed the levels of these cytokines produced by CD4⁺CD25^– cells. However, CD4⁺CD25⁺ PBL produced higher levels of IL‐4 and similar amounts of IL‐10 as CD4⁺CD25^– cells. Regulatory CD4⁺CD25⁺ T cells have an activated phenotype in the thymus with expression of CTLA‐4 and CD122 (IL‐2Rβ). The fact that CD4⁺CD25⁺ regulatory T cells are present with a similar frequency in the thymus of humans, ratsand mice, suggests that the role of these cells in the maintenance of immunological tolerance is an evolutionarily conserved mechanism.