Transmissible spongiform encephalopathies (TSEs) are neurodegenerative disorders characterized by the accumulation in the CNS of a pathological conformer (PrPTSE) of the host-encoded cellular prion protein (PrPC). PrPTSE has a central role in the pathogenesis of the disease but other factors are likely involved in the pathological process. In this work we employed a multi-step proteomic approach for the identification of proteins that co-purify with the protease-resistant core of PrPTSE (PrP27-30) extracted from brains of hamsters with experimental scrapie. We identified ferritin, calcium/calmodulin-dependent protein kinase α type II, apolipoprotein E, and tubulin as the major components associated with PrP27-30 but also trace amounts of actin, cofilin, Hsp90α, the γ subunit of the T-complex protein 1, glyceraldehyde 3-phosphate dehydrogenase, histones, and keratins. Whereas some of these proteins (tubulin and ferritin) are known to bind PrP, other proteins (calcium/calmodulin-dependent protein kinase α type II, Hsp90α) may associate with PrPTSE fibrils during disease. Apolipoprotein E and actin have been previously observed in association with PrPTSE, whereas cofilin and actin were shown to form abnormal rods in the brain of patients with Alzheimer disease. The roles of these proteins in the development of brain lesions are still unclear and further work is needed to explain their involvement in the pathogenesis of TSEs.