Multiple lines of evidence suggest that CD4⁺ lymphocytes initiate autoimmune responses against myelin antigens in multiple sclerosis (MS). The increased frequency of activated myelin-specific cells in MS patients indicates that the activation of autoreactive cells represents a central event in the pathogenesis of the disease. We identified a CD4⁺ subpopulation that is characterized phenotypically by the persistent absence of surface CD28 expression and functionally by CD28-independent activation and Th1 cytokine secretion. Owing to their costimulation-independent activation and their expression of a full agonist signaling activation pattern, CD4⁺CD28^– cells have the potential to initiate autoimmune responses in the central nervous system, a compartment devoid of professional antigen presenting cells. Long-term memory CD4⁺CD28^– cells produce high amounts of IFN-γ and maximally upregulate IFN-γ and IL-12Rβ2 chain expression in the absence of costimulation. They exhibit prominent growth characteristics and increased survival after activation, likely related to their persistent lack of CTLA-4 surface expression. The CD4⁺CD28^– population is expanded in a subgroup of MS patients. Myelin basic protein-specific cells detected in this cell subset may play an important role in the inflammatory response within the central nervous system.