We have previously shown that conventional as well as germ-free CD4+ T cells depleted of CD25+ cells from the gut-associated lymphoid tissue and the periphery proliferate specifically in response to enterobacterial antigen exposure whereas unfractionated CD4+ T cells are not reactive under these conditions. Here we show that the majority of the enteroantigen-specific CD4+CD25− T cells are naive cells expressing a CD45RBhigh, CD62Lhigh and CD44low phenotype. These cells are also present in the thymus and data from adult thymectomized mice show that they represent late (>6 weeks) thymic emigrants. Upon enteroantigen activation, the CD4+CD25− T cells secrete IL-4, IL-5, IL-10, granulocyte macrophage colony-stimulating factor, tumor necrosis factor-α and IFN-γ. Clonotype mapping of the TCRBV regions 1–18 of enteroantigen-reactive CD4+CD25− T cells by TCR clonotype mapping revealed the polyclonal nature of this subset. In conclusion, we have for the first time demonstrated the presence of an evolutionary, functionally conserved subset of CD4+ T cells, which are reactive against enterobacterial antigens. This subset resides both in the thymus and the periphery; it is not dependent on previous antigen experience and represents late thymic emigrants, which by enteroantigen-induced activation express a mixed Th1–Th2 phenotype. At homeostatic conditions, CD25+ T cells maintain peripheral tolerance in this CD4+ T cell subset.