Experimental autoimmune encephalomyelitis (EAE) is a widely adopted animal model system for studying human multiple sclerosis that affects the central nervous system (CNS). To understand the underlying pathogenic mechanisms of the autoimmune T cell response, localization, enumeration and characterization of autoreactive T cells are essential. We assessed encephalitogenic proteolipid protein epitope (PLP(139-151))-specific T cells in the periphery and CNS of SJL/J mice using MHC class II I-As multimers during both pre-clinical and clinical phases of PLP-induced EAE in conjunction with T cell function. Our results strongly suggest that PLP(139-151)-specific CD4+ T cells first expand primarily in the CNS-draining cervical lymph nodes and then migrate to the CNS. In the CNS, these PLP-specific CD4+ T cells accumulate, become activated and differentiate into effector cells that produce IFN-gamma in response to the self-peptide.