OBJECTIVE: To evaluate the efficacy and safety of repeated rituximab treatment based on the assessment of peripheral circulating memory B cells over 24 months in patients with relapsing neuromyelitis optica (NMO). DESIGN: Prospective open-label study. SETTING: Institutional referral center for multiple sclerosis. Patients  Thirty patients with relapsing NMO or NMO spectrum disorder. Intervention  Treatment protocol of rituximab consisted of an induction therapy (375 mg/m(2) once weekly for 4 weeks or 1000 mg infused twice, with a 2-week interval between the infusions) followed by maintenance therapy. The maintenance therapy was repeated treatment with rituximab (375 mg/m(2), once) whenever the frequency of reemerging CD27(+) memory B cells was more than 0.05% in peripheral blood mononuclear cells by flow cytometric analysis. MAIN OUTCOME MEASURES: Annualized relapse rate, disability (Expanded Disability Status Scale score), anti-aquaporin 4 antibody level, and safety of rituximab treatment. RESULTS: Of 30 patients, 28 showed a marked reduction in relapse rate while taking rituximab over 24 months. The relapse rate was reduced significantly, by 88%, and 70% of patients became relapse-free over 24 months. Disability either improved or stabilized in 97% of patients. Anti-aquaporin 4 antibody levels declined significantly following treatment with rituximab, consistent with the clinical response and the effect on CD27(+) memory B cells. Repeated treatment with rituximab was generally well tolerated, and no clinically relevant adverse event leading to discontinuation of treatment was observed. CONCLUSION: Repeated treatment with rituximab appeared to produce consistent and sustained efficacy over 24 months with good tolerability in patients with NMO.