It has been suggested that mucosally induced tolerance could be elicited by selective activation of T helper (Th) cell subsets. We have directly investigated the role of Th 1 and Th2-type cells in mucosally induced tolerance using IFN-γ and IL-4 knockout (-/-) mice. The IFN-y-/- and IL-4-/- and normal mice were given 25 mg of ovalbumin (OVA) before systemic immunization with OVA in CFA. Normal mice had significantly suppressed DTH responses, and lower OVA-specific serum IgG including IgGl, IgG2a and IgG2b subclasses when compared with mice fed PBS only before systemic immunization. These immune responses were accompanied by a marked reduction of OVA-specific T cell proliferation. Further, when Thland Th2-type cytokines were examined by ELISA in culture supernatants from OVA-stimulated spleen cells from tolerized mice, significantly lower levels of IFN-y, IL-2, IL-4, IL-5 and IL-6 were detected in comparison to non-tolerized normal mice. When IL-4''- mice were orally immunized in an identical manner, a state of oral tolerance was induced. On the other hand, oral immunization of the IFN-f' mice with OVA resulted in normal DTH responses and OVA-specific T cell proliferation. Thus, mucosally mediated tolerance was not induced in IFN-y/' mice. These findings suggest that IFN-Y is a potent cytokine for induction of T cell mediated systemic unresponsiveness.