CD8⁺CD25⁺ cells, which expressed high levels of Foxp3, glucocorticoid-induced tumor necrosis factor receptor (GITR), CCR8, tumor necrosis factor receptor 2 (TNFR2), and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) mRNAs, were identified in the fibrous septa and medullary areas of human thymus. Activated CD8⁺CD25⁺ thymocytes did not produce cytokines, but most of them expressed surface CTLA-4 and transforming growth factor β1 (TGF-β1). Like CD4⁺CD25⁺, CD8⁺CD25⁺ thymocytes suppressed the proliferation of autologous CD25-T cells via a contact-dependent mechanism. The suppressive activity of CD8⁺CD25⁺ thymocytes was abrogated by a mixture of anti-CTLA-4 and anti-TGF-β1 antibodies and it was mediated by their ability to inhibit the expression of the interleukin 2 receptor α chain on target T cells. These results demonstrate the existence of a subset of human CD8⁺CD25⁺ thymocytes sharing phenotype, functional features, and mechanism of action with CD4⁺CD25⁺ T regulatory cells. (Blood. 2003;102:4107-4114)