Homing behavior and function of autoimmune CD4⁺ T cells in vivo was analyzed before and during EAE, using MBP-specific T cells retrovirally engineered to express the gene of green fluorescent protein. The cells migrate from parathymic lymph nodes to blood and to the spleen. Preceding disease onset, large numbers of effector cells invade the CNS, with only negligible numbers left in the periphery. In early EAE, most (>90%) infiltrating CD4⁺ cells were effector cells. Migratory effector cells downregulate activation markers (CD25, OX-40) but upregulate several chemokine receptors and adsorb MHC class II on their membranes. Within the CNS, the effector cells are reactivated, with upregulated proinflammatory cytokines and downmodulated T cell receptor-associated structures, presumably reflecting autoantigen recognition in situ.