Clonally expanded CD8⁺ T lymphocytes are present in multiple sclerosis lesions, as well as in the cerebrospinal fluid of patients with multiple sclerosis. In experimental autoimmune encephalomyelitis, CD8⁺ T lymphocytes are found in spinal cord and brainstem lesions. However, the exact phenotype of central nervous system-infiltrating CD8⁺ T lymphocytes and the mechanism by which these cells cross the blood–brain barrier remain largely unknown. Using cerebrospinal fluid from patients with multiple sclerosis, spinal cord from experimental autoimmune encephalomyelitis and coronavirus-induced encephalitis, we demonstrate that central nervous system-infiltrating CD8⁺ T lymphocytes are mostly of the effector memory phenotype (CD62L⁻ CCR7⁻ granzymeB^hi). We further show that purified human effector memory CD8⁺ T lymphocytes transmigrate more readily across blood-brain barrier-endothelial cells than non-effector memory CD8⁺ T lymphocytes, and that blood-brain barrier endothelium promotes the selective recruitment of effector memory CD8⁺ T lymphocytes. Furthermore, we provide evidence for the recruitment of interferon-γ- and interleukin-17-secreting CD8⁺ T lymphocytes by human and mouse blood-brain barrier endothelium. Finally, we show that in vitro migration of CD8⁺ T lymphocytes across blood-brain barrier-endothelial cells is dependent on α4 integrin, but independent of intercellular adhesion molecule-1/leucocyte function-associated antigen-1, activated leucocyte cell adhesion molecule/CD6 and the chemokine monocyte chemotactic protein-1/CCL2. We also demonstrate that in vivo neutralization of very late antigen-4 restricts central nervous system infiltration of CD8⁺ T lymphocytes in active immunization and adoptive transfer experimental autoimmune encephalomyelitis, and in coronavirus-induced encephalitis. Our study thus demonstrates an active role of the blood-brain barrier in the recruitment of effector memory CD8⁺ T lymphocytes to the CNS compartment and defines α4 integrin as a major contributor of CD8⁺ T lymphocyte entry into the brain.