The mucosal immune system is uniquely adapted to elicit immune responses against pathogens but also to induce tolerogenic responses to harmless antigens. In mice, nasal application of ovalbumin (OVA) leads to suppression of both Th1 and Th2 responses. This tolerance can be transferred to naive mice by CD4+ Tr cells from the spleen. Using the allotypic Ly5 system, we were able to demonstrate in vivo that Tr cells not only suppress naive CD4+ T cells, but also induce them to differentiate into Tr cells. The effector function of these mucosal Tr cells is not restricted by cytokine polarization, since Tr cells from Th1-tolerant mice can suppress a Th2 response and vice versa. Transfer of splenic CD4+CD25+ and CD4+CD25- T cell subsets from OVA-tolerized mice revealed that both subsets were equally able to suppress a delayed-type hypersensitivity response in acceptor mice. In contrast to the CD25- T cell subset, the CD25+ cells were not specific for the antigen used for tolerization. Together, these findings demonstrate a role for CD4+CD25- Tr cells in mucosal tolerance, which suppresses CD4+ T cells in an antigen-specific fashion, irrespective of initial Th1/Th2 skewing of the immune response. This offers a major advantage in the manipulation of mucosal tolerance for the treatment of highly cytokine-polarized disorders such as asthma and autoimmune diseases.