CD25⁺4⁺ regulatory T cells (T_reg) play an indispensable role in preventing autoimmunity. Little is known, however, about the antigen specificities required for their development and effector functions. Mice transgenic for an anti-myelin basic protein (MBP) T cell antigen receptor (TCR) spontaneously develop experimental autoimmune encephalomyelitis (EAE) when deficient for the RAG-1 gene (T/R⁻), whereas RAG-1-competent transgenic animals (T/R⁺) remain healthy, protected by CD4⁺ T_reg-expressing endogenous TCRs. We have now investigated the role and specificity of CD25⁺4⁺ T_reg in this system. The results show that T/R⁺ animals contain MBP-specific suppressive CD25⁺4⁺ cells, whereas T/R⁻ do not. Adoptive transfer of CD25⁺4⁺ cells from nontransgenic or T/R⁺ donors into T/R⁻ mice prevented the development of EAE. Surprisingly, transfer of nontransgenic CD25⁺4⁺ cells purified from T/R⁺ donors conferred only a limited protection, possibly because of their restricted repertoire diversity that we demonstrate here. Absence of transgenic CD25⁺4⁺ cells in animals deficient for endogenous TCRα chains and analyses of endogenous TCR gene expression in subsets of CD4⁺ cells from T/R⁺ mice demonstrate that development of transgenic MBP-specific CD25⁺4⁺ T_reg depends on the coexpression of endogenous TCRα chains. Taken together, these results indicate that specificity to MBP is required for effector functions but is not sufficient for thymic selection/commitment of CD25⁺4⁺ T_reg preventing EAE.