SJL mice are highly susceptible to experimental autoimmune encephalomyelitis (EAE) induced with myelin proteolipid protein (PLP) peptide 139-151, whereas H-2 congenic B10.S mice are resistant. Immunodominance and susceptibility to EAE are associated with a high precursor frequency of PLP 139-151-specific T cells in the naïve repertoire of SJL mice. To understand the mechanism of EAE resistance in B10.S mice, we determined the precursor frequency of PLP 139-151-reactive T cells in both strains by using IA^s/PLP 139-151 tetramers. SJL and B10.S mice had similar frequencies of tetramer-reactive T cells in the naïve peripheral repertoire. However, in SJL mice, the majority of PLP 139-151 tetramer-positive cells were in the CD4⁺CD25^- population, whereas there were more tetramer-positive cells in the CD4⁺CD25⁺ population of B10.S mice. Depletion of CD4⁺CD25⁺ cells in vivo facilitated the expansion of PLP 139-151-reactive cells with production of T helper 1 cytokines in EAE-resistant B10.S mice. Furthermore, anti-CD25 Ab treatment before immunization resulted in EAE induction in these otherwise resistant mice. These data indicate an important role for autoantigen-specific CD4⁺CD25⁺ cells in genetic resistance to autoimmunity.