Abstract Background The monoclonal antibody natalizumab (NAT) inhibits the migration of lymphocytes throughout the bloodâ brain barrier by blocking very late antigen (VLA)-4 interactions, thereby reducing inflammatory central nervous system (CNS) activity in patients with multiple sclerosis (MS). We evaluated the effects of different NAT treatment regimens. Methods We developed and optimised a NAT assay to measure free NAT, cell-bound NAT and VLA-4 expression levels in blood and cerebrospinal fluid (CSF) of patients using standard and prolonged treatment intervals and after the cessation of therapy. Results In paired CSF and blood samples of NAT-treated MS patients, NAT concentrations in CSF were approximately 100-fold lower than those in serum. Cell-bound NAT and mean VLA-4 expression levels in CSF were comparable with those in blood. After the cessation of therapy, the kinetics of free NAT, cell-bound NAT and VLA-4 expression levels differed. Prolonged intervals greater than 4Â weeks between infusions caused a gradual reduction of free and cell-bound NAT concentrations. Sera from patients with and without NAT-neutralising antibodies could be identified in a blinded assessment. The NAT-neutralising antibodies removed NAT from the cell surface in vivo and in vitro. Intercellular NAT exchange was detected in vitro. Conclusions Incorporating assays to measure free and cell-bound NAT into clinical practice can help to determine the optimal individual NATÂ dosing regimen for patients with MS.