Scrapie is a transmissible spongiform encephalopathy of sheep and other mammals in which disease appears to be caused by the accumulation of an abnormal form of a host protein, prion protein (PrP), in the brain and other tissues. The process by which the normal protease-sensitive form of PrP is converted into the abnormal protease-resistant form is unknown. Several hypotheses predict that oligomeric forms of either the normal or abnormal PrP may act as intermediates in the conversion process. We have now identified a 60-kDa PrP derived from hamster PrP expressed in murine neuroblastoma cells. Peptide mapping studies provided evidence that the 60-kDa PrP was composed solely of PrP and, based on its molecular mass, appeared to be a PrP dimer. The 60-kDa PrP was not dissociated under several harsh denaturing conditions, which indicated that it was covalently linked. It was similar to the disease-associated form of PrP in that it formed large aggregates. However, it resembled the normal form of PrP in that it was sensitive to proteinase K and had a short metabolic half-life. The 60-kDa PrP, therefore, had characteristics of both the normal and disease-associated forms of PrP. Formation and aggregation of the 60-kDa hamster PrP occurs in uninfected mouse neuroblastoma cells, which suggests that hamster PrP has a predisposition to aggregate even in the absence of scrapie infectivity. Similar 60-kDa PrP bands were identified in scrapie-infected hamster brain but not in uninfected brain. Therefore, a 60-kDa molecule might participate in the scrapie-associated conversion of protease-sensitive PrP to protease-resistant PrP.