Thymus-derived regulatory T lymphocytes of CD4⁺CD25⁺ phenotype regulate a large variety of beneficial and deleterious immune responses and can inhibit lethal graft-versus-host disease in rodents. In vitro, CD4⁺CD25⁺ T cells require specific major histocompatibility complex (MHC)/peptide ligands for their activation, but once activated they act in an antigen-nonspecific manner. In vivo, regulatory T cells are also activated in an antigen-specific fashion, but nothing is known about antigen specificity of their suppressor-effector function. Here we show that CD4⁺CD25⁺ regulatory T lymphocytes isolated from naive mice and activated in vitro with allogeneic antigen-presenting cells (APCs) induced specific long-term tolerance to bone marrow grafts disparate for major and minor histocompatibility antigens; whereas “target” bone marrow was protected, third-party bone marrow was rejected. Importantly, in mice injected with a mix of target and third-party bone marrows, protection and rejection processes took place simultaneously. These results indicate that CD4⁺CD25⁺ regulatory T cells can act in an antigen-specific manner in vivo. Our results suggest that CD4⁺CD25⁺ regulatory T cells could in the future be used in clinical settings to induce specific immunosuppression. (Blood. 2004;103:4216-4221)