Leukocyte trafficking from the blood to local inflammatory sites is essential for the initiation and maintenance of tissue specific immune responses. In autoimmune diseases such as multiple sclerosis (MS), leukocyte transmigration from the blood to the target organ is dependent on intercellular cell adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expressed by the endothelial cells (ECs). In this study we describe CD166/activated leukocyte cell adhesion molecule (ALCAM) as a novel adhesion molecule of the human blood–brain barrier (BBB). We demonstrate that inflammatory cytokines up-regulate the expression of ALCAM on the surface of BBB-ECs in vitro and that endothelial ALCAM co-localizes with leukocyte expressed-CD6 in the transmigratory cup during migration. We further show that ALCAM blocking antibody restricts the transmigration of CD4, CD14 and CD19 immune cells across human BBB-ECs and that ALCAM expression is increased on ECs within active MS lesions, as compared to normal appearing white matter and to non-MS brains. These results suggest an important role for ALCAM in the recruitment of leukocytes to the CNS and identify ALCAM as a potential target to modulate CNS inflammatory reactions.