Interferon beta (IFNbeta) is a first-line therapy for multiple sclerosis (MS). However, some patients experience a decline in efficacy with continued therapy due to the development of anti-IFNbeta neutralizing antibodies (NAb). We investigated the frequency of NAb cross-sectionally in 846 MS patients who were receiving IFNbeta-1b, IFNbeta-1a im, or IFNbeta-1a sc. The frequency of NAb in patients receiving IFNbeta-1a im was lower (5%) than in patients treated with any other form of IFNbeta (22-35%) (P < 0.0001). Binding antibodies (BAb) were measured in 808 patients. The frequency differed significantly between treatment groups, ranging from 45% (IFNbeta-1a im) to 88% (IFNbeta-1b). The proportion of NAb-positive patients within the BAb-positive group differed significantly among treatment groups, ranging between 12% (IFNbeta-1a im) and 51% (IFNbeta-1a sc). The median NAb titer from all IFNbeta-1a-treated patients was higher than from IFNbeta-1b-treated patients (446 versus 171 NU/ mL, P = 0.04). Among NAb-positive patients, the frequency of NAb titers > 100 NU/mL was 71% for IFNbeta-1a compared with 58% for IFNbeta-1b (P = 0.04). Except for conflicting data regarding IFNbeta-1a sc, the results are generally consistent with the literature and together with the differing proportion of NAb-positive patients within the BAb-positive group, provide further insight into the immunogenicity of the IFNbeta preparations.