Clonally expanded populations of B cells carrying somatic mutations of Ig variable (V) region genes have been detected in the CNS of subjects with multiple sclerosis (MS), suggesting that a process of B cell affinity maturation with ensuing production of potentially pathogenic autoantibodies may occur inside the CNS. Here, we have characterized the B cell subsets present in the cerebrospinal fluid (CSF) of MS patients and of individuals with other inflammatory neurological disorders by flow cytometry. CD19(+)CD38(high+)CD77(+), Ki67(+), Bcl-2(-) centroblasts, i.e., a B cell subset found exclusively in secondary lymphoid organs, were detected in the CSF but not in paired peripheral blood from both patient groups. CD27(+)IgD(-) memory B cells, i.e., cells with hyper-mutated IgV genes, were significantly increased in the CSF vs. paired peripheral blood and displayed up-regulation of the CD80 and CD86 costimulatory molecules and of CC chemokine receptor (CCR) 1, CCR2, and CCR4 in both patient groups. Lymphotoxin-alpha, CXC ligand (CXCL) 12, and CXCL13, key mediators of lymphoid neogenesis, were present in the CSF from patients with MS and other inflammatory neurological disorders and were expressed in MS brain tissue, with selective localization in the outer layer of the capillary vessel wall. In conclusion, this study suggests that a compartmentalized B cell response occurs within the CNS during an ongoing inflammatory reaction, through a recapitulation of all stages of B cell differentiation observed in secondary lymphoid organs. The presence of lymphotoxin-alpha, CXCL12, and CXCL13 in the CNS may provide favorable microenvironmental conditions for these events.