A new recombinant vesicular stomatitis virus (rVSV) that expresses green fluorescent protein (GFP) on the cytoplasmic domain of the VSV glycoprotein (G protein) was used in the mouse as a model for studying brain infections by a member of the Mononegavirales order that can cause permanent changes in behavior. After nasal administration, virus moved down the olfactory nerve, first to periglomerular cells, then past the mitral cell layer to granule cells, and finally to the subventricular zone. Eight days postinoculation, rVSV was eliminated from the olfactory bulb. Little sign of infection could be found outside the olfactory system, suggesting that anterograde or retrograde axonal transport of rVSV was an unlikely mechanism for movement of rVSV out of the bulb. When administered intracerebrally by microinjection, rVSV spread rapidly within the brain, with strong infection at the site of injection and at some specific periventricular regions of the brain, including the dorsal raphe, locus coeruleus, and midline thalamus; the ventricular system may play a key role in rapid rVSV dispersion within the brain. Thus, the lack of VSV movement out of the olfactory system was not due to the absence of potential for infections in other brain regions. In cultures of both mouse and human central nervous system (CNS) cells, rVSV inoculations resulted in productive infection, expression of the G-GFP fusion protein in the dendritic and somatic plasma membrane, and death of all neurons and glia, as detected by ethidium homodimer nuclear staining. Although considered a neurotropic virus, rVSV also infected heart, skin, and kidney cells in dispersed cultures. rVSV showed a preference for immature neurons in vitro, as shown by enhanced viral infection in developing hippocampal cultures and in the outer granule cell layer in slices of developing cerebellum. Together, these data suggest a relative affinity of rVSV for some neuronal types in the CNS, adding to our understanding of the long-lasting changes in rodent behavior found after transient VSV infection.