Infection of the central nervous system (CNS) is a defining feature of scrapie. Several findings suggest that scrapie agent invades the CNS via the splanchnic and vagus nerve after ingestion of infectivity. Here we address the involvement of the enteric nervous system (ENS) and gut-associated lymphoid tissue (GALT) in this pathogenetic process. Immunocytochemistry was used for the detection of pathological PrP in the duodenum and ileum of hamsters fed with 263K scrapie and sacrificed at different stages of incubation. The experiments revealed early infection of various GALT components and of submucosal and myenteric ENS ganglia. These results provide evidence for an important role of the ENS in scrapie neuroinvasion and for centripetal vagal spread of infection from the gut to the brain after oral uptake of agent.