Whether regulatory T cells could suppress the immune responses to an unrelated Ag and could induce tolerance to that Ag was investigated. In female B6 (I-Ab) mice tolerized to MHC class II I-Abm12 (bm12) alloantigen by cyclophosphamide-induced system, regulatory T cells specific for bm12 Ag were induced. These regulatory T cells suppressed in vivo immune responses to an unrelated Ag (male Ag) that was co-expressed with bm12 Ag on the same graft. However, the immune responses to male Ag suppressed by regulatory T cells were reverted by immunization with male Ag alone, indicating regulatory T cells suppressed the induction of immune responses to the unrelated Ag co-expressed with suppressogenic determinant, but did not inactivate T cells specific for that third Ag, such as clonal anergy or deletion. These results provided that minor Ag adjacent to major Ag, even if they were expressed on the professional APC, could be suppressed in the periphery, and the responses to those minor Ag could be activated when the suppression mechanism was abrogated or ineffective.