BACKGROUND AND PURPOSE: Neutralizing antibodies (NAbs) appearing during treatment with Interferon-beta (IFN-beta) reduce or abolish bioactivity and therapeutic efficacy. Initial combination therapy with methylprednisolone (MP) may reduce the frequency of NAb positive patients. We hypothesized that MP treatment might also reduce NAb levels and re-establish IFN-beta bioactivity in patients already NAb+, who discontinue IFN-beta therapy. METHODS: In a 6-month open-label trial, we compared monthly high-dose pulsed MP treatment in 38 Nab positive patients with 35 NAb+, MP-untreated control patients discontinuing any therapy or switching to glatiramer acetate. All patients were NAb+ with an absent in vivo response to IFN-beta. NAbs were measured using a cytopathic effect assay and expressed as neutralizing capacity (NC) in percentage of added IFN-beta. Bioactivity was expressed as in vivo Myxovirus Resistance Protein A (MxA) mRNA induction in whole blood using real time PCR. RESULTS: At the end of study, median NAb NC was 92% in both groups. Eight patients (21%) in the MP group and four patients (11%) in the control group had regained an in vivo MxA response to IFN-beta (P = 0.35). CONCLUSIONS: Monthly pulsed MP treatment in NAb positive patients has no beneficial effect on NAb status or IFN-beta bioactivity.