To elucidate the role of CD4⁺CD25⁺ regulatory T cells in oral tolerance, we used the model of contact hypersensitivity (CHS) to 2,4-dinitrofluorobenzene (DNFB), which is mediated by CD8⁺ Tc1 effector cells independently of CD4⁺ T-cell help. Conversely to normal mice, invariant chain knock-out (KO) (Ii^°/°) mice, which are deficient in CD4⁺ T cells, cannot be orally tolerized and develop a chronic hapten-specific CHS response. Transfer of naive CD4⁺ T cells before hapten gavage into Ii^°/° mice restores oral tolerance by a mechanism independent of interleukin-10 (IL-10) production by CD4⁺ T cells. That naturally occurring CD4⁺CD25⁺ T cells are critical for oral tolerance induction is demonstrated by the finding that (1) transfer of CD4⁺CD25⁺ but not CD4⁺CD25^– T cells into Ii^°/° recipients completely prevents the CHS response and skin infiltration by CD8⁺ T cells, by blocking development of hapten-specific CD8⁺ T cells; (2) in vivo depletion of CD4⁺CD25⁺ cells by antibody treatment in normal mice impairs oral tolerance; and (3) CD4⁺CD25⁺ T cells inhibit hapten-specific CD8⁺ T-cell proliferation and interferon γ (IFNγ) production, in vitro. These data show that naturally occurring CD4⁺CD25⁺ T cells are instrumental for orally induced tolerance and are key actors for the control of antigen-specific CD8⁺ T-cell effectors mediating skin inflammation.