T cell receptor engagement and the B7‐CD28 / CTLA‐4 signaling pathways play critical roles in T cell activation and regulation. CD28 engagement results in T cell activation, differentiation and survival while CTLA‐4 signals block IL‐2 production, cell cycle progression and T cell differentiation. We explored the role of CTLA‐4 in peripheral tolerance induced by intravenous administrationof ethylene carbodiimide‐fixed, antigen‐coupled splenocytes in the PLP139 – 151‐induced relapsing experimental autoimmune encephalomyelitis system. Tolerance induction with PLP139 – 151‐coupled splenocytes correlates with low B7 expression on the fixed antigen‐presenting cells, conditions that would favor CTLA‐4‐mediated inhibition. Administration of CTLA‐4Ig or anti‐CTLA‐4 concomitant with the ‘tolerogenic’ stimulus, however, failed to reverse tolerance induction. In contrast, blocking CTLA‐4 at the time of secondary ‘immunogenic’ encounter with antigen reversed the tolerant state. These findings indicate that CTLA‐4 is required to maintain the unresponsive state of the tolerized T cells upon antigenic stimulation under inflammatory conditions and, therefore, have important implications for therapeutic regulation of autoimmune disease.