Background: B cells are implicated in the pathogenesis of multiple sclerosis. A beneficial effect of B-cell depletion using rituximab has been shown, but the complete mechanism of action for this drug is unclear. Objective: To determine the relationship between T and B cells and changes in cerebrospinal fluid (CSF) chemokine levels with rituximab, a monoclonal antibody that targets CD20. Design: Phase 2 trial of rituximab as an add-on therapy. Setting: The John L. Trotter Multiple Sclerosis Center, Washington University. Participants and Intervention: Thirty subjects who had relapsing-remitting multiple sclerosis with clinical and magnetic resonance imaging activity despite treatment with an immunomodulatory drug received 4 weekly doses of rituximab (375 mg/m2). Main Outcome Measures: Lumbar puncture was performed before and after rituximab infusions in 26 subjects. Levels of B and T lymphocytes in the CSF were enumerated by flow cytometry, and chemoattractant levels were measured by enzyme-linked immunosorbent assay. Results: After rituximab administration, CSF B-cell levels were decreased or undetectable in all subjects, and CSF T-cell levels were reduced in 21 subjects (81%). The mean reduction in CSF cellularity was 95% for B cells and 50% for T cells. After rituximab infusion, CSF CXCL13 and CCL19 levels decreased (P=.002 and P=.03, respectively). The proportional decline in CSF T-cell levels correlated with the proportional decrease in CXCL13 levels (r=0.45; P=.03), suggesting a possible relationship. The CSF IgG index, IgG concentration, and oligoclonal band number were unchanged following treatment. Conclusions: In subjects with multiple sclerosis, B cells are critical for T-cell trafficking into the central nervous system and may alter the process by influencing chemokine production within the central nervous system.