Thymectomy of BALB/c mice on day 3 of life results in the development of autoimmune gastritis (AIG) due to the absence of CD4+ CD25+ regulatory T cells. However, depletion of CD4+ CD25+ T cells by treatment with anti-CD25 rarely resulted in AIG. Depletion was efficient, as transfer of splenocytes from depleted mice induced AIG in nu/nu mice. One explanation for this result is that CD4+ CD25− T cells upon transfer to nude recipients undergo lymphopenia-induced proliferation, providing a signal for T cell activation. Cotransfer of CD25+ T cells did not inhibit initial proliferation but did suppress AIG. Surprisingly, immunization with the AIG target Ag, H/K ATPase, in IFA failed to induce disease in normal animals but induced severe AIG in CD25-depleted mice. These results demonstrate that second signals (nonspecific proliferation, TCR activation, or inflammation) are needed for induction of autoimmunity in the absence of CD25+ regulatory T cells.