The chemokines CXCL9 and CXCL10 bind to the common receptor CXCR3 and are implicated in the pathogenesis of T‐cell‐mediated immunity in the central nervous system (CNS). Here we examined the temporal and spatial regulation of the Cxcl9 and Cxcl10 genes in the CNS of mice with myelin oligodendrocyte glycoprotein (MOG)‐induced experimental autoimmune encephalomyelitis (EAE) and by glial cells in vitro. During peak disease the levels of CXCL9 and CXCL10 mRNA and protein were increased significantly in the cerebellum and spinal cord but were reduced during the recovery phase. Expression of these genes in the CNS was abolished in IFN‐γ‐receptor deficient mice with MOG‐EAE. In wild‐type mice, CXCL9 RNA was localized mainly to infiltrating mononuclear cells including lesion and perilesional microglia, while CXCL10 RNA was seen primarily in more distal astrocytes that surrounded the inflammatory lesions. Examination of cultured glia following treatment with IFN‐γ revealed that while both CXCL9 and CXCL10 mRNA transcripts were induced in microglia, only CXCL10 mRNA was induced in astrocytes. Thus, although IFN‐γ is the pivotal mediator of both Cxcl10 and Cxcl9 gene expression in EAE, this cytokine differentially regulates the expression of these genes by astrocytes and microglia. The differential glial localization of these chemokines in EAE suggests CXCL9 and CXCL10 have specialized functions. © 2007 Wiley‐Liss, Inc.