Astroglial proliferation associated with pathological prion protein (PrPsc) deposition is widely described in Transmissible Spongiform Encephalopathies (TSEs). However, little is known of the actual role played by glia in their pathogenesis. The aim of the study has been to determine whether PrPsc is located exclusively in neurons or in both neurons and glial cells present in the central nervous system in a natural Scrapie model. Samples of cerebellum from 25 Scrapie sheep from various flocks were sectioned. Following epitope retrieval with formic acid, proteinase K and heat treatment, primary antibody L42 and primary antibodies against glial fibrillary acidic protein were applied as prion- and astrocytic-specific markers, respectively. For visualization, a suitable mixture of fluorochrome-conjugated secondary antibodies was used. Relevant controls were processed in the same manner. As determined by confocal microscopy, PrPsc deposits co-localized with glial cells in all samples. Our results suggest that these cells can sustain active prion propagation, in agreement with similar findings from other studies of primary cell cultures and inoculated mice. Furthermore, despite ongoing debate regarding whether varied TSE sources show differences in their tropism for different cell lineages in the brains of affected animals, no differences in co-localization results were seen.