Experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats and the bromodeoxyuridine (BrdU)-incorporating cells were demonstrated immunohistochemically in lesions in the central nervous system (CNS) to assess the extent of T cell proliferation during EAE. In active EAE, BrdU+ cells were most numerous on day 12 postimmunization, when both clinical signs and inflammation detected by histologic examination were most severe; they declined thereafter although a considerable number of inflammatory foci remained in some rats. In passive EAE, BrdU+ cells were most numerous 2 days before the full-blown EAE and then rapidly decreased in number. On day 6 post-transfer, the CNS showed the most severe histologic changes but virtually no inflammatory cells in the lesions were labeled with BrdU. Double immunofluorescence staining with T cell (OX52) and macrophage/microglia (OX42) markers showed that about half of the BrdU+ cells were labeled with OX52 at the peak of EAE. The proportion of BrdU+ OX52+ T cells at later stages was about 20%. BrdU+ OX42+ cells ranged between 50 and 80% throughout the course of the disease. Furthermore, serial pulsing experiments during passive EAE revealed that inflammatory cells that had been labeled with BrdU outside the CNS and found later in the CNS were 15 times more numerous than those labeled in situ in the CNS. Taken together, these findings indicate that the majority of T cells involved in EAE undergo DNA synthesis outside the CNS and then infiltrate into the CNS, and that T cells labeled in situ in the CNS are few, and decrease rapidly in number. Since interleukin-2-receptor-positive cells detected by mAb OX39 outnumbered BrdU+ T cells at these later stages, we postulate that an unresponsive state instead of T-cell proliferation was induced in the CNS.