To study the role of CD25(+) regulatory T cells (T(regs)) in peripheral B cell tolerance, we generated transgenic rat insulin promoter RIP-OVA/HEL mice expressing the model Ags OVA and HEL in pancreatic islet beta cells (where RIP is rat insulin promoter and HEL is hen egg lysozyme). Adoptively transferred transgenic OVA-specific CD4(+) and CD8(+) T cells proliferated only in the autoantigen-draining pancreatic lymph node (PLN), demonstrating pancreas-specific Ag expression. Transferred HEL-specific transgenic B cells (IgHEL cells) disappeared within 3 wk from transgenic but not from nontransgenic mice immunized with autoantigen. Depletion of CD25(+) FoxP3(+) cells completely restored IgHEL cell numbers. T(reg) exerted an analogous suppressive effect on endogenous HEL-specific autoreactive B cells. T(regs) acted by inhibiting the proliferation of IgHEL cells in the spleen and PLN and by systemic induction of their apoptosis. Furthermore, they reduced BCR and MHC II surface expression on IgHEL cells in the PLN. These findings demonstrate that autoreactive B cells specific for a nonlymphoid tissue autoantigen are controlled by T(regs).