Dynamic interplay between cytokines and chemokines directs trafficking of leukocyte subpopulations to tissues in autoimmune inflammation. We have examined the role of IFN-γ in directing chemokine production and leukocyte infiltration to the CNS in experimental autoimmune encephalomyelitis (EAE). BALB/c and C57BL/6 mice are resistant to induction of EAE by immunization with myelin basic protein. However, IFN-γ-deficient (BALB/c) and IFN-γR-deficient (C57BL/6) mice developed rapidly progressing lethal disease. Widespread demyelination and disseminated leukocytic infiltration of spinal cord were seen, unlike the focal perivascular infiltrates in SJL/J mice. Gr-1+ neutrophils predominated in CNS, and CD4+ T cells with an activated (CD69+, CD25+) phenotype and eosinophils were also present. RANTES and macrophage chemoattractant protein-1, normally up-regulated in EAE, were undetectable in IFN-γ-and IFN-γR-deficient mice. Macrophage inflammatory protein-2 and T cell activation gene-3, both neutrophil-attracting chemokines, were strongly up-regulated. There was no induction of the Th2 cytokines, IL-4, IL-10, or IL-13. RNase protection assays and RT-PCR showed the prevalence of IL-2, IL-3, and IL-15, but no increase in IL-12p40 mRNA levels in IFN-γ-or IFN-γR-deficient mice with EAE. Lymph node cells from IFN-γ-deficient mice proliferated in response to myelin basic protein, whereas BALB/c lymph node cells did not. These findings show a regulatory role for IFN-γ in EAE, acting on T cell proliferation and directing chemokine production, with profound implications for the onset and progression of disease.