Technical Abstract: In the western United States, biting midges (Culicoides spp.) have been implicated as possible insect vectors for vesicular stomatitis virus (VSV), which causes economically important disease outbreaks in cattle and horses. Information regarding the interaction of VSV within its insect vectors is limited. Within any competent vector, virus obtained from a blood meal must be able to penetrate the midgut barrier, infect the midgut epithelium, replicate, and disseminate to epidemiologically significant organs such as the salivary glands and ovaries. This extensive infection must occur without negatively affecting the health or feeding behavior of the transmitting insect. In vivo infection studies were performed in adult females to examine sites of VSV replication, with respect to vector competence, and to determine the progression of viral infection within this insect vector. VSV was able to escape the midgut barrier, disseminate quickly and replicate in epithelial, neural and hemolymph cells throughout the insect, as evidenced by RT-PCR, in situ hybridization and transmission electron microscopy. Additionally, time course immunohistochemical analysis revealed digestive, circulatory, and neural infection pathways. This is the first whole-body, microscopic analysis of a putative arthropod-borne virus in this known insect vector.