Induction of peripheral tolerance by steady-state peripheral dendritic cells (DCs) presenting self antigen may be important in preventing autoimmune diseases mediated by self-reactive T cells that escape thymic deletion. However, the relative contribution of thymic and peripheral tolerance to the inactivation of self-specific repertoires is yet to be clearly defined. Here we tested the relative contribution of thymic and peripheral tolerance induction, using mice (11c.OVA) in which ovalbumin (OVA) expression is genetically targeted to DCs in conjunction with mice (Vbeta5 TCR transgenic), where a polyclonal repertoire of OVA-specific T cells of diverse affinity is present. The expression of OVA in thymic DC reduced the frequency of OVA(257-264)-specific mature CD8 single-positive thymocytes although some functional OVA-specific CD8(+) T cells escaped negative selection and were detectable in the periphery. After adult thymectomy, OVA(257-264)-reactive T cells declined in the periphery indicating that the repertoire of OVA(257-264)-specific T cells that escaped negative selection and egressed to the periphery, were susceptible to inactivation by steady-state peripheral DC. Thus, in the face of inefficient negative selection, peripheral tolerance induction to cognate antigen by resting DC is a crucial requirement for the inactivation of a self-specific repertoire.