BACKGROUND Neutralizing antibodies (NAbs) to IFN-beta may have a detrimental effect on treatment response, but increasing IFN-beta dose could reduce their occurrence. The OPTimization of Interferon for MS (OPTIMS) study was a multicenter trial investigating clinical and MRI outcomes with the approved IFN-beta-1b dose (250 microg) and a higher dose (375 microg), s.c. every other day. OBJECTIVE To analyze the occurrence of NAbs and their effect on clinical and MRI response over a long-term (4-year) follow-up using cross-sectional and longitudinal statistical analysis. METHODS Relapses or disease progression was assessed open-label and MRI scans were performed serially during the first year of the study. Neutralizing antibodies were measured using the MxA protein production neutralization assay. RESULTS A total of 145 patients with relapsing-remitting multiple sclerosis from 14 centers participated in the study. Neutralizing antibody frequency was negatively associated with MRI treatment response, but no detrimental effect of NAbs on the clinical response was observed. Results obtained using cross-sectional or longitudinal statistical approaches were similar. Over the 4-year period, NAb-positive patients treated with 375 microg had a significantly greater probability of NAb disappearance (hazard ratio: 3.41; 95% confidence interval: 1.78 - 6.43; p < 0.01). CONCLUSION Use of an IFN-beta-1b dose higher than the currently approved 250-microg dose is associated with an increased probability of NAb disappearance. The OPTIMS study was registered at ClinicalTrials.gov: NCT00473213.