The role of various adhesion molecules in lymphocyte homing to the brain and in inflammatory autoimmune disease of the central nervous system (CNS) was examined in mice. Activated T cell lines and clones expressed CD44 and integrin alpha4, but not L-selectin, and entered the CNS independent of their antigen specificity. mAbs directed against CD44 and integrin alpha4 prevented the transfer of experimental autoimmune encephalomyelitis (EAE) by myelin basic protein-specific T cells. T cells preincubated with anti-CD44 or antiintegrin alpha4 were blocked only partially from entering the brain parenchyma. However, both antibodies efficiently prevented CNS inflammation and clinical expression of EAE when injected in vivo. This effect lasted as long as antibodies were administered. Antibodies specific for L-selectin had no effect on homing of encephalitogenic T cells to the brain or development of EAE. Antiintegrin alpha4 and anti-CD44 did not impair the activation and function of encephalitogenic T cells in vitro and did not deplete integrin alpha4- or CD44-positive cells in vivo. These data suggest that, in the absence of leukocyte recruitment, the entry of a reduced number of activated myelin basic protein-reactive T cells in the CNS is not sufficient for the development and expression of EAE. We propose that antibodies to integrin alpha4 and CD44 prevent clinical disease by partially targeting the primary influx of encephalitogenic T cells and by preventing the secondary influx of leukocytes to lesions initiated by the transferred T cells.