CD4+ CD25+ regulatory T cells prevent organ-specific autoimmune diseases in various animal models. We analysed human lymphoid tissues to identify similar CD25+ regulatory T cells. Adult peripheral blood contained two populations of CD4+ T cells that expressed CD25 at different densities. The larger population (≈ 40%) expressed intermediate levels of CD25 (CD25+) and displayed a memory T-cell phenotype (CD45RA−/RO+, CD45RBlow, CD95+, CD62Llow, CD38low). The smaller population of cells (≈ 2%) expressed very high levels of CD25 (CD25++). In addition to the activation/memory T-cell antigens mentioned above they also expressed intracellular CD152 (CTLA-4) as well as enhanced levels of cell-surface CD122, similar to the murine CD4+ CD25+ regulatory counterpart. To exclude that the CD25++ cells had not been recently primed by external antigen we analysed cord blood and thymus. CD25++, CD152+ and CD122++ cells were present in paediatric thymus (10% of CD4+ CD8− thymocytes) expressing signs of recent selection (CD69+) and in cord blood (5% of CD4+ cells) where they showed a naive phenotype. In addition, cord blood contained a small population of CD25+ cells (≈ 2% of CD4 T cells) that were CD152− and CD122low and displayed signs of activation. Together with published data that CD25+ CD25++ cells from the thymus and peripheral blood are regulatory, our results suggest that regulatory CD25+ T cells leave the thymus in a naïve state and become activated in the periphery.