To investigate whether the multifocal inflammatory disease in TGFβ1-deficient mice is caused by self-antigen (self-Ag)-specific autoreactive T cells, or whether it is caused by antigen independent, spontaneous hyperactivation of T cells, we have generated Tgfb1 −/− and Tgfb1 −/− Rag1 −/− mice expressing the chicken OVA-specific TCR transgene (DO11.10). On a Rag1-sufficient background, Tgfb1 −/− DO11.10 mice develop a milder inflammation than do Tgfb1 −/− mice, and their T cells display a less activated phenotype. The lower level of activation correlates with the expression of hybrid TCR (transgenic TCRβ and endogenous TCRα), which could recognize self-Ag and undergo activation. In the complete absence of self-Ag recognition (Tgfb1 −/− DO11.10 Rag1 −/− mice) inflammation and T-cell activation are eliminated, demonstrating that self-Ag recognition is required for the hyper-responsiveness of TGFβ1-deficient T cells. Thus, TGFβ1 is required for the prevention of autoimmune disease through its ability to control the activation of autoreactive T cells to self-Ag.