Transgenic mice expressing chimeric prion protein (PrP) genes derived from Syrian hamster (SHa) and mouse (Mo) PrP genes were constructed. One SHa/MoPrP gene, designated MH2M PrP, contains five amino acid substitutions encoded by SHaPrP, while another construct, designated MHM2 PrP, has two substitutions. Transgenic (Tg) (MH2M PrP) mice were susceptible to both Syrian hamster and mouse prions, whereas three lines expressing MHM2 PrP were resistant to Syrian hamster prions. The brains of Tg(MH2M PrP) mice dying of scrapie contained chimeric PrPSc and prions with an artificial host range favoring propagation in mice that express the corresponding chimeric PrP and were also transmissible, at reduced efficiency, to nontransgenic mice and hamsters. Our findings provide genetic evidence for homophilic interactions between PrPSc in the inoculum and PrPc synthesized by the host.