Injection of antigen into the thymus of adult animals induces specific systemic tolerance, but the mechanisms of acquired thymic tolerance are not well understood. To investigate these mechanisms we used a model of intrathymic injection of ovalbumin (OVA) in BALB/c mice. We show an antigen-specific decrease in proliferative responses to OVA, as well as a significant decrease in antigen-specific IL-2 secretion and IFN-γ production by splenocytes and lymph node cells of tolerant mice. Addition of recombinant IL-2in vitroreversed the defect in IFN-γ production by cells from OVA-tolerized animals, but did not reverse the proliferation or IL-2 production defects. By using an adoptive transfer system, where a small population of OVA peptide-specific CD4 + TCR transgenic T cells are transferred into syngeneic normal recipients, we show an absence of peripheral antigen-dependent clonal expansion of transferred CD4 + TCR transgenic cells in tolerant micein vivo.There was an increase in clonotype-positive T cells in the thymus after immunization, confirming that activated T cells circulate through the thymus. Furthermore, thymectomy after intrathymic injection abrogates the effect of acquired thymic tolerance and restores antigen-dependent clonal expansionin vivo.We conclude that intrathymic injection of antigen induces Th1 cell unresponsiveness and prevents the peripheral expansion of antigen-specific CD4 + T cellsin vivo.This is the first demonstration that in acquired thymic tolerance antigen-specific T cells circulate to the thymus where they may be anergized or ultimately deleted.