Objectives: To confirm that neutralizing antibodies (NAb) to interferon beta can persist after therapy withdrawal and to evaluate whether persisting NAb are associated with a worse clinical disease course in multiple sclerosis (MS). Design: Retrospective study. Setting: Tertiary referral center in the Netherlands. Patients: A total of 71 patients with relapsing-remitting multiple sclerosis treated with interferon beta in the past. Main Outcome Measures: Persisting NAb after therapy withdrawal were tested using the cytopathic effect assay. Patients with and without persisting NAb were compared on several outcomes: the change in annualized relapse rate from prior to interferon beta treatment initiation to after cessation of treatment, time to sustained disability on the Kurtzke Expanded Disability Status Scale, and the use of disease-modifying treatments after cessation of treatment with interferon beta. Results: Seventeen of 71 patients (24%) tested NAb positive after a median interval of 25 months (interquartile range, 10-51 months) after interferon beta treatment cessation. Eleven of these 17 patients (15%) were hightiter NAb positive (>150 10-fold reduction units per mL). Persisting NAb were associated with an increase in the annualized relapse rate (P=.04) and a reduction in time to reach a sustained Expanded Disability Status Scale score of 6.0, ie, the need for unilateral assistance to walk 100 m(P=.02). Moreover, NAb-positive patients were treated with second-line therapy significantly more often, especially mitoxantrone (P=.006). Conclusion: Anti-interferon beta NAb can persist after interferon beta treatment withdrawal and are associated with overt clinical disease activity. This is made apparent by an increase in relapse rate and faster disability progression and is supported by the observed need for more aggressive therapy after interferon beta treatment cessation. Prospective studies are warranted to confirm these results. ©2010 American Medical Association. All rights reserved.