Recent evidence suggests that co-stimulation provided by B7 molecules through CTLA-4 is important in establishing peripheral tolerance. In the present study, we examined the kinetics of tolerance induction and T cell differentiation following i.p. administration of myelin basic protein (MBP) Ac1-11 in mice transgenic for a TCR V(beta)8.2 gene derived from an encephalitogenic T cell clone specific for MBP Ac1-11. Examination of the lymph node cell response after antigen administration demonstrated a dependence on CTLA-4 for i.p. tolerance induction. Examination of splenocyte responses suggested that i.p. antigen administration induced a T(h)2 response, which was potentiated by anti-CTLA-4 administration. Interestingly, i.p. tolerance was able to inhibit the induction of experimental autoimmune encephalomyelitis and anti-CTLA-4 administration did not alter this phenotype, suggesting that CTLA-4 blockade did not block tolerance induction. Thus, T cell differentiation and the dependence on CTLA-4 for tolerance induction following i.p. antigen administration differs between lymph node and spleen in a model of organ-specific autoimmunity.