The 'protein-only' hypothesis of prion propagation argues that infectious prions consist of PrP(Sc), a conformational isomer of host-derived prion protein (PrP(C)), which can be distinguished from PrP(C) by its partial resistance to proteases. While protease-resistant PrP has been produced by mixing PrP(Sc) and recombinant-derived PrP(C) in vitro, bioassay of any new infectivity has been precluded by the need to use a large molar excess of same species PrP(Sc). Transgenic mice expressing a chimaeric hamster-mouse PrPC (MH2M PrP(C)) are, unlike conventional mice, highly susceptible to Sc237 hamster scrapie. In addition, they produce MH2M PrP(Sc) and infectivity which is pathogenic for conventional mice. We have therefore attempted to produce MH2M PrP(Sc) in vitro as any infectivity produced could be distinguished from the hamster PrP(Sc) used to promote the conversion by bioassay in conventional mice. Although protease-resistant MH2M PrP was produced, no infectivity was detected on bioassay. These results argue that acquisition of protease resistance by PrP(C) is not sufficient for the propagation of infectivity.