Type I interferons (IFN) are crucial in host defense but also are implicated as causative factors for neurological disease. Interferon regulatory factor (IRF9) is involved in type I IFN-regulated gene expression where it associates with STAT1:STAT2 heterodimers to form the transcriptional complex ISGF3. The role of IRF9 in cellular responses to type I IFN is poorly definedin vivoand hence was examined here. While transgenic mice (termedGIFN) with chronic production of low levels of IFN-α in the CNS were relatively unaffected, the same animals lacking IRF9 [GIFNxIRF9 knock-out(KO)] had cataracts, became moribund, and died prematurely. The brain ofGIFNxIRF9 KOmice showed calcification with pronounced inflammation and neurodegeneration whereas inflammation and retinal degeneration affected the eyes. In addition, IFN-γ-like gene expression in the CNS in association with IFN-γ mRNA and increased phosphotyrosine-STAT1 suggested a role for IFN-γ. However,GIFNxIRF9 KOmice deficient for IFN-γ signaling developed an even more severe and accelerated disease, indicating that IFN-γ was protective. In IRF9-deficient cultured mixed glial cells, IFN-α induced prolonged activation of STAT1 and STAT2 and induced the expression of IFN-γ-like genes. We conclude that (1) type I IFN signaling and cellular responses can occurin vivoin the absence of IRF9, (2) IRF9 protects against the pathophysiological actions of type I IFN in the CNS, and (3) STAT1 and possibly STAT2 participate in alternative IRF9-independent signaling pathways activated by IFN-α in glial cells resulting in enhanced IFN-γ-like responses.