Relapsing experimental autoimmune encephalomyelitis (R-EAE) induced with the immunodominant epitope from proteolipid protein, PLP 139–151, is characterized by the development of recurrent relapses with recruitment of T cells reactive to additional myelin peptides, including PLP 179–191 (epitope spreading). In this study, we have determined that the CD28/B7 costimulatory pathway is involved in this process. We found preferential up-regulation of B7-1 during the course of R-EAE and a selective increase in its functional costimulatory activity, relative to B7-2. Anti B7-1 F(ab) fragment therapy, but not anti B7-2 MAb therapy, blocked clinical relapses, ameliorated CNS pathology, and blocked epitope spreading. These results suggest that the maintenance of autoimmune reactivity in EAE depends on CD28/B7-1-dependent costimulation of newly recruited T cells responsible for epitope spreading. These studies have important implications for the role of epitope spreading in disease progression and the clinical application of costimulatory antagonists in autoimmune diseases.