During the pathogenesis of multiple sclerosis (MS), activated B-cells cross the inflamed endothelium of the central nervous system (CNS) to exert their effector functions, probably associated with the action of several adhesion molecules. B-cell mobilization towards the CNS already occurs after the first demyelinating events suggestive of MS, known as clinically isolated syndrome (CIS). However, little is known about the role of these adhesion molecules at this early disease stage. We, therefore, evaluated the relationship between the expression of α4 integrin by peripheral B-cell subsets and disease activity. We found that α4 integrin was up-regulated in all B-cell subsets from patients with CIS and that its expression was correlated with the number of gadolinium-enhanced lesions. A comparison of B-cell subsets distribution in the CSF of patients with CIS, of patients with other inflammatory neurological diseases (OIND) and of patients with non-inflammatory neurological diseases (NIND) showed that the percentages of CSF CD19(+) B-cells were significantly higher in the CIS and OIND group. In addition, CIS and OIND CSF were enriched in switched and MZ-like memory B-cells, although all B-cell subsets were present. These results suggest that up-regulation of α4 integrin may enhance B-cell accumulation within the CSF at the time of CIS.