Scrapie and Creutzfeldt-Jakob disease are transmissable, degenerative neurological diseases caused by prions. Considerable evidence argues that prions contain protease-resistant sialoglycoproteins, designated PrP(Sc), encoded by a cellular gene. The prion protein (PrP) gene also encodes a normal cellular protein designated PrP(C). We established clonal cell lines which support the replication of mouse scrapie or Creutzfeldt-Jakob disease prions. Mouse neuroblastoma N2a cells were exposed to mouse scrapie prions and subsequently cloned. After limited proteinase K digestion, three PrP-immunoreactive proteins with apparent molecular masses ranging between 20 and 30 kilodaltons were detected in extracts of scrapie-infected N2a cells by Western (immuno-) blotting. The authenticity of these PrP(Sc) molecules was established by using monospecivic antiserum raised against a synthetic peptide corresponding to a portion of the prion protein. Those clones synthesizing PrP(Sc) molecules possessed scrapie prion infectivity as measured by bioassay; clones without PrP(Sc) failed to demonstrate infectivity. Detection of PrP(Sc) molecules in scrapie-infected N2a cells supports the contention that PrP(Sc) is a component of the infectious scrapie particle and opens new approaches to the study of prion diseases.