The development of somatically mutated memory and plasma B cells is a consequence of T cell-dependent antigen-challenged humoral immunity. To investigate the role of B cell-mediated humoral immunity in the initiation and evolution of multiple sclerosis (MS), we analyzed Ig variable heavy chain genes of intrathecal B cells derived from patients with a first clinical manifestation suggestive of MS. Sequences of Ig variable regions showed that B cells in the cerebrospinal fluid from most of these patients were clonally expanded and carried somatic hypermutated variable heavy chain genes. The mutations showed a high replacement-to-silent ratio and were distributed in a way suggesting that these clonally expanded B cells had been positively selected through their antigen receptor. In comparison, intrathecal B-cell clonal expansion often precedes both oligoclonal IgG bands and multiple magnetic resonance imaging lesions. Clinical follow-up study showed that patients with clonally expanded intrathecal B cells had a high rate of conversion to clinically definite MS. The findings provide direct evidence of recruitment of germinal center differentiated B lymphocytes into the central nervous system during the initiation of MS. These results indicate B cell-mediated immune response in the cerebrospinal fluid is an early event of inflammatory reaction in the central nervous system of MS. This procedure also provides a more sensitive method to evaluate the association of humoral immunity in the evolution of MS.