B7/CD28-mediated costimulation is a promising target for therapeutic intervention in autoimmune diseases. However, studies addressing the differential functional roles of B7-1 and B7-2 in several autoimmune models have resulted in conflicting data, perhaps due to the temporal dynamics of B7-1 and B7-2 surface expression on different cell types and/or at different sites during an autoimmune response. We examined the temporal expression of B7 costimulatory molecules in the CNS and in various lymphoid organs during the course of murine relapsing-remitting experimental autoimmune encephalomyelitis (R-EAE). Following immunization of SJL mice with the immunodominant proteolipid protein epitope, PLP139–151, surface expression of B7-1 was up-regulated on B cells, T cells, and macrophages, relative to B7-2, on CNS-infiltrating cells and on splenocytes. Similar enhancement in splenic B7-1 expression could be induced in SJL mice by the adoptive transfer of PLP139–151-specific cells or by immunization with CFA alone. These changes were not observed on lymph node cells, including those isolated from lymph nodes draining the immunization site, which maintained the predominant B7-2 expression pattern seen in naive mice. These phenotypic expression patterns correlated with the functional predominance of B7-1 in costimulating T cell activation when employing APCs from the spleen or CNS of mice with ongoing R-EAE, while B7-2 remained functionally predominant on lymph node APCs. Variation of phenotypic expression and functional dominance of costimulatory molecule expression in different lymphoid compartments during an active inflammatory autoimmune response has important implications in immune regulation, autoimmune pathogenesis, and therapeutic strategies.