Background and purpose: Infection with the John Cunningham virus (JCV) is required for the development of progressive multifocal leukoencephalopathy, the feared complication of natalizumab treatment in multiple sclerosis patients. The JCV seroconversion rate seems higher in natalizumab treated patients than in the normal population, with an unknown cause. Methods: Natalizumab concentration was correlated to JCV antibody status and seroconversion in a large cohort of multiple sclerosis patients. Results: One hundred and thirty-five patients were included. No correlation was found between natalizumab concentration and JCV status, JCV seroconversion or JCV index. Conclusions: Higher natalizumab concentrations do not explain the increased JCV seroconversion rate in natalizumab treated patients.