Multiple sclerosis (MS) is an inflammatory disease of the myelinated central nervous system that is postulated to be induced by myelin-reactive CD4 T cells. T cell activation requires an antigen-specific signal through the TCR and a costimulatory signal, which can be mediated by B7–1 or B7–2 engagement of CD28. To directly examine the activation state of myelin-reactive T cells in MS, the costimulation requirements necessary to activate myelin basic protein (MBP) or tetanus toxoid (TT)-reactive CD4 T cells were compared between normal controls and MS patients. Peripheral blood T cells were stimulated with Chinese hamster ovary (CHO) cells transfected either with DRB1* 1501/DRA0101 chains (t-DR2) alone, or in combination with, B7–1 or B7–2. In the absence of costimulation, T cells from normal subjects stimulated with the recall antigen TT p830–843 were induced to expand and proliferate, but stimulation with MBP p85–99 did not have this effect. In marked contrast, T cells from patients with MS stimulated with MBP p85–99 in the absence of B7–1 or B7–2 signals expanded and proliferated. Thus, MBP-reactive CD4 T cells in patients with MS are costimulation independent and have been previously activated in vivo. These experiments provide further direct evidence for a role of activated MBP-specific CD4 T cells in the pathogenesis of MS.