In experimental allergic encephalomyelitis (EAE), autoimmune T cells infiltrate the central nervous system (CNS) and initiate demyelinating pathology. We have used flow cytometry to directly analyse the migration to the CNS of MBP-reactive CD4+ T cells labelled with a lipophilic fluorescent dye (PKH2), in SJL/J mice with passively transferred EAE. Labelled cells constituted about 45% of the CNS CD4+ population at the time of EAE onset. Almost all (greater than 90%) of the PKH2-labelled CD4+ T cells from EAE CNS were blasts and were alpha/beta T cell receptor (TCR)+, CD44(Pgp-1)high, and the majority were CD45RB(low). By contrast, most PKH2-labelled CD4+ T cells in lymph nodes, although CD44high, were CD45RBhigh cells. The cells that were transferred to induce EAE were essentially similar to antigen-primed lymph node cell populations, containing less than 15% CD44high cells, and most of them were CD45RBhigh. The CD44high CD45RB(low) phenotype is characteristic of memory/effector T cells that have been activated by antigen recognition. The difference in CD45RB expression between CNS and LN could therefore reflect differential exposure and/or response to antigen. Consistent with this, PKH2-labelled CD4+ cells isolated from the CNS were responsive to MBP in vitro, whereas PKH2+ CD4+ cells from lymph nodes showed almost undetectable responses. In control experiments in which ovalbumin (OVA)-reactive T cells were transferred, a small number of fluorescent-labelled CD4+ T cells were also detected in CNS, but there were very few blasts, and these remained CD45RBhigh. These results argue for induction of the memory/effector phenotype of CD4+ T cells, and their selective retention in the CNS, as a consequence of antigen recognition.