Antigen injection into the thymus of adult animals induces systemic tolerance and protects animals from subsequent challenge for the autoimmune disease. However, its mechanisms are not well understood. In this study, we analyzed tolerance to experimental autoimmune encephalomyelitis (EAE) induced in Lewis rats by intrathymic (i.t.) injection of myelin basic protein (MBP). Intrathymic injection of MBP 7 days before immunization with MBP/complete Freund's adjuvant resulted in complete suppression of clinical signs of EAE in most animals and markedly reduced the histological severity in the central nervous system lesion. However, immunohistochemical examination and the TCR repertoire analysis revealed that there was no significant difference in the T cell composition in the lesion and the TCR spectratype pattern between MBP and saline i.t. rats, suggesting that encephalitogenic T cell activation occurs equally in both protected and symptomatic rats. In contrast, quantitative analysis of cytokine mRNA and flow cytometry revealed a marked increase of IL-10 production in the splenic macrophages/dendritic cell (Mø/DC) population of MBP i.t. rats. Adoptive transfer of this population significantly suppressed the clinical course of EAE in recipients. Taken together, IL-10-secreting Mø/DC in peripheral lymphoid organs activated by MBP i.t. injection may play a critical role in the induction and maintenance of tolerance.