Relapsing experimental autoimmune encephalomyelitis (R-EAE) induced with the immunodominant epitope from proteolipid protein, PLPIJo-151, ischaracterized by the development of recurrent relapses with recruitment of T cells reactive to additional myelin peptides, including PLP178-101 (epitope spreading). In this study, we have determined that the CD2g/B7 costimulatory pathway is involved in this process. We found preferential up-regulation of 87-l during the course of R-EAE and a selective increase in its functional costimulatory activity, relative to 87-2. Anti B7-1 F (ab) fragment therapy, but not anti 87-2 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-l-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.