To study B-cell tolerance against non-lymphoid tissue autoantigens, we generated transgenic rat insulin promoter (RIP)-OVA/hen egg lysozyme (HEL) mice expressing the model antigens, OVA and HEL, in pancreatic islets. Their vaccination with OVA or HEL induced far less auto-Ab titers compared with non-transgenic controls. Depletion of CD25(+) cells during immunization completely restored auto-Ab production, but did not affect antibodies against a foreign control antigen. Depletion at later time-points was not effective. OVA-specific CD25(+) FoxP3(+) T(reg) were more frequent in the autoantigen-draining pancreatic LN than in other secondary lymphatics of RIP-OVA/HEL mice. Consistently, B cells were suppressed in that LN and also in the spleen, which is known to concentrate circulating antigen, such as the antigens used for vaccination. Suppression involved preventing expansion of autoreactive B cells in response to autoantigen, reducing antibody production per B-cell and isotype changes. These findings demonstrate that CD25(+) T(reg) suppress auto-Ab production against non-lymphoid tissue antigens in an antigen-specific manner.