Early events in multiple sclerosis (MS) lesion formation are loss of blood–brain barrier (BBB) integrity, immune cell trafficking into the central nervous system, and demyelination. To date, the molecular mechanisms underlying these pathogenic events are poorly understood. Heparin‐binding epidermal growth factor (HB‐EGF) is a trophic factor that is induced by inflammatory stimuli and has previously been shown to interact with tetraspanins (TSPs), a family of transmembrane proteins that are involved in cellular migration and adhesion. Given the known roles of TSPs and HB‐EGF, we hypothesized that HB‐EGF and TSPs may play a role in the processes that underlie MS lesion formation. We examined the expression of HB‐EGF and the TSPs CD9 and CD81 in MS brain and found that HB‐EGF was highly induced in reactive astrocytes in active lesions. TSPs were constitutively expressed throughout normal appearing white matter and control white matter. In contrast, CD9 was reduced in demyelinated lesions and increased on blood vessels in lesion areas.In vitrostudies revealed that expression of HB‐EGF and TSPs is regulated during inflammation. Importantly, blocking either HB‐EGF or CD9 significantly reduced the migration of monocytes across brain endothelial cell monolayers. Moreover, blocking CD9 strongly enhanced the barrier function of the BBBin vitro. Together, we demonstrate that these molecules are likely implicated in processes that are highly relevant for MS lesion formation, and therefore, HB‐EGF and TSPs are promising therapeutic targets. GLIA 2013;61:1890–1905