Regulatory CD4⁺CD25⁺ T cells (Treg) are mandatory for maintaining immunologic self-tolerance. We demonstrate that the cell-cell contact–mediated suppression of conventional CD4⁺ T cells by human CD25⁺ Treg cells is fixation resistant, independent from membrane-bound TGF-β but requires activation and protein synthesis of CD25⁺ Treg cells. Coactivation of CD25⁺ Treg cells with Treg cell–depleted CD4⁺ T cells results in anergized CD4⁺ T cells that in turn inhibit the activation of conventional, freshly isolated CD4⁺ T helper (Th) cells. This infectious suppressive activity, transferred from CD25⁺ Treg cells via cell contact, is cell contact–independent and partially mediated by soluble transforming growth factor (TGF)-β. The induction of suppressive properties in conventional CD4⁺ Th cells represents a mechanism underlying the phenomenon of infectious tolerance. This explains previously published conflicting data on the role of TGF-β in CD25⁺ Treg cell–induced immunosuppression.