In view of the established link between bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease and of the susceptibility of sheep to experimental BSE, the detection of potential cases of naturally occurring BSE in sheep has become of great importance. In this study, the immunohistochemical (IHC) phenotype of disease-associated prion protein (PrP) accumulation has been determined in the brain of 64 sheep, of various breeds and PrP genotypes, that had developed neurological disease after experimental BSE challenge with different inocula by a range of routes. Sheep BSE was characterized by neuron-associated intra- and extracellular PrP aggregates and by conspicuous and consistent deposits in the cytoplasm of microglia-like cells. The stellate PrP type was also prominent in most brain areas and marked linear deposits in the striatum and midbrain were distinctive. Sheep of the ARR/ARR and ARQ/AHQ genotypes displayed lower levels of PrP than other sheep, and intracerebral BSE challenge resulted in higher levels of PrP accumulating in the brain compared with other routes. The PrP genotype and the route of challenge also appeared to affect the incubation period of the disease, giving rise to complex combinations of magnitude of PrP accumulation and incubation period. Despite these differences, the phenotype of PrP accumulation was found to be very consistent across the different factors tested (notably after subpassage of BSE in sheep), thus highlighting the importance of detailed IHC examination of the brain of clinically affected sheep for the identification of potential naturally occurring ovine BSE.