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Phenotypic markers, localization, functional activities, and mechanisms of action in vitro of CD4⁺CD25⁺ T cells, purified from postnatal human thymuses, were investigated. These cells showed poor or no proliferation in mixed lymphocyte culture (MLC), and suppressed in a dose-dependent fashion the proliferative response to allogeneic stimulation of CD4⁺CD25⁻ thymocytes. Virtually all CD4⁺CD25⁺ thymocytes constitutively expressed cytoplasmic T lymphocyte antigen (CTLA)-4, surface tumor necrosis factor type 2 receptor (TNFR2), and CCR8. They prevalently localized to perivascular areas of fibrous septa and responded to the chemoattractant activity of CCL1/I-309, which was found to be produced by either thymic medullary macrophages or fibrous septa epithelial cells. After polyclonal activation, CD4⁺CD25⁺ thymocytes did not produce the cytokines interleukin (IL)-2, IL-4, IL-5, IL-13, interferon γ, and only a …