Transcription of vesicular stomatitis virus is controlled by the position of a gene relative to the single 3′ genomic promoter: promoter-proximal genes are transcribed at higher levels than those in more 5′ distal positions. In previous work, we generated viruses having rearranged gene orders. These viruses had the promoter-proximal gene that encodes the nucleocapsid protein, N, moved to the second or fourth position in the genome in combination with the glycoprotein gene, G, moved from its usual promoter-distal fourth position to the first or third position. This resulted in three new viruses identified by the positions of the N and G genes in the gene order: G3N4, G1N4, and G1N2. The viruses G3N4 and G1N4 were attenuated for lethality in mice. In the present study, we addressed the basis of this attenuation by measuring the ability of each of the rearranged viruses to travel to and replicate in the olfactory bulb and brain following intranasal inoculation. In addition, the neuropathogenicity, serum cytokine levels, and immunoglobulin G isotype profiles in infected mice were determined. All the viruses reached the olfactory bulb and brain, but the outcomes of these infections were dramatically different. Viruses N1G4(wt) and G1N2 caused lethal encephalitis in 100% of animals within 7 days postinoculation; however, viruses G3N4 and G1N4 were cleared from the brain by 7 days postinoculation and all animals survived without apparent distress. The viruses differed in the distribution and intensity of lesions produced and the type and levels of cytokines induced. Animals inoculated with N1G4(wt) or G1N2 displayed extensive encephalitis and meningitis and had elevated levels of serum gamma interferon compared to what was seen with G3N4- or G1N4-infected mice. In contrast to what occurred with intranasal inoculation, all four viruses caused lethal encephalitis when administered by direct inoculation to the brain, a route that circumvents the majority of the host immune response, demonstrating that G3N4 and G1N4 were not deficient in their abilities to cause disease in the brain. These findings indicate that gene rearrangement and its consequent alteration of gene expression can, without any other changes, alter the viral spread and cytokine response following intranasal infection.