Neuromyelitis optica (NMO) is characterized by selective inflammation of the spinal cord and optic nerves but is distinct from multiple sclerosis (MS). Interferon (IFN)-β mitigates disease activity in MS, but is controversial in NMO, with a few reports of disease worsening after IFN-β therapy in this highly active disease. In systemic lupus erythematosus (SLE), IFNs adversely affect disease activity. This study examines for the first time whether serum IFN-α/β activity and IFN-β-induced responses in peripheral blood mononuclear cells (MNC) are abnormally elevated in NMO, as they are in SLE, but contrast to low levels in MS.Serum type I IFN-α/β activity was measured by a previously validated bioassay of 3 IFN-stimulated genes (RT-PCR sensitivity, 0.1 U/ml) rather than ELISA, which has lower sensitivity and specificity for measuring serum IFNs. IFN responses in PBMNC were assessed by in vitro IFN-β-induced activation of phospho-tyrosine-STAT1 and phospho-serine-STAT1 transcription factors, and MxA proteins using Western blots.Serum IFN-α/β activity was highest in SLE patients, followed by healthy subjects and NMO, but was surprisingly low in therapy-naïve MS. In functional assays in vitro, IFN-β-induced high levels of P-S-STAT1 in NMO and SLE, but not in MS and controls. IFN-β-induced MxA protein levels were elevated in NMO and SLE compared to MS.Serum IFN activity and IFN-β-induced responses in PBMNC are elevated in SLE and NMO patients versus MS. This argues for similarities in pathophysiology between NMO and SLE and provides an explanation for IFN-induced disease worsening in NMO.