Development of neutralizing antibodies (NAbs) reduces the clinical efficacy of interferon beta (IFNbeta) treatment in multiple sclerosis (MS) patients. The aim of this study was to evaluate NAb seroprevalence (frequency of patients with NAbs) and immunogenicity (titer levels) of IFNbeta preparations in a clinical setting. We analysed 1115 consecutive MS patients, treated with one of the three available IFNbeta preparations, for an average of 40 months (1-120 months), for the presence of NAbs with the MxA protein induction assay. Overall, 32% of patients were positive for NAbs with neutralizing titers above 10. The frequency of NAbs, ie, the seroprevalence, was 13% in Avonex-treated patients, 43% for Betaferon, 39% for Rebif22 and 30% for Rebif44. In addition, the potential to induce high titer levels, ie, the immunogenicity, was observed to differ between preparations. Avonex, showing the lowest seroprevalence, also showed low immunogenicity and typically induced low titers. Betaferon, showing the highest seroprevalence when inducing NAbs, induced lower titers compared to Rebif22 and Rebif44. Treatment duration over five years only marginally correlated with decreased seroprevalence and titer levels. In conclusion, NAbs to IFNbeta are common in a clinical setting and the IFNbeta preparations differ not only in NAb seroprevalence, but also in immunogenicity.