By applying immunohistochemistry and employing conditional reporter mice that express firefly luciferase under control of the IFN-β promoter in either all or only distinct cell types, we found that astrocytes are the main producers of IFN-β after infection of the brain with diverse neurotropic viruses, including rabies virus, Theiler’s murine encephalomyelitis virus and vesicular stomatitis virus. This finding was surprising given the fact that neurons are the principal targets of all these viruses. Analysis of a panel of knockout mouse strains revealed that sensing of viral components via both RIG-I-like helicases and Toll-like receptors contributes to IFN induction in the virus-infected brain. When using an in vivo approach to permanently mark infected cells, we observed that a substantial number of astrocytes must have transiently encountered virus during early stages of the infection. Thus, our data strongly indicate that abortive viral infection of astrocytes triggers pattern-recognition receptor signalling, which results in secretion of IFN-β.