Nearly 100% of NOD. H-2h4 mice develop spontaneous autoimmune thyroiditis (SAT) and produce anti-mouse thyroglobulin autoantibodies when they receive 0.05% NaI in their drinking water beginning at 8 wk of age. Our previous studies showed that TGFβ1 mRNA was constitutively expressed in thyroids and spleens of normal NOD. H-2h4 mice but not other strains of mice. To determine whether TGFβ might have a role in SAT, mice were given anti-TGFβ mAb at various times during development of SAT. Anti-TGFβ markedly inhibited development of SAT and production of anti-mouse thyroglobulin IgG1 autoantibodies. Anti-TGFβ was most effective in inhibiting SAT when given during the time thyroid lesions were developing, ie, starting 4 wk after administration of NaI water. The active form of the TGFβ1 protein was present in thyroids of mice with SAT but not in normal NOD. H-2h4 thyroids. However, thyrocytes of normal NOD. H-2h4 thyroids did express latent TGFβ1. TGFβ1 protein expression in the thyroid correlated with SAT severity scores, and administration of anti-TGFβ inhibited TGFβ1 protein expression in both the thyroid and spleen. TGFβ1 was produced primarily by inflammatory cells and was primarily localized in areas of the thyroid containing clusters of CD4+ T and B cells. Depletion of CD8+ T cells had no effect on TGFβ1 protein expression. Activation of splenic T cells was apparently not inhibited by anti-TGFβ, because up-regulation of mRNA for cytokines and other T cell activation markers was similar for control and anti-TGFβ-treated mice. TGFβ1 may function by promoting migration to, or retention of, inflammatory cells in the thyroid.