Early studies in transgenic mouse lines have shown that the coexpression of endogenous murine prion protein (PrPC) and transgenic PrPC from another species either inhibits or allows the propagation of prions, depending on the infecting prion strain and interacting protein species. The way whereby this phenomenon, so-called "interference," is modulated remains to be determined. In this study, different transgenic mouse lines were crossbred to produce mice coexpressing bovine and porcine PrPC, bovine and murine PrPC, or murine and porcine PrPC These animals and their respective hemizygous controls were inoculated with several prion strains from different sources (cattle, mice, and pigs) to examine the effects of the simultaneous presence of PrPC from two different species. Our results indicate interference with the infection process, manifested as extended survival times and reduced attack rates. The interference with the infectious process was reduced or absent when the potentiality interfering PrPC species was efficiently converted by the inoculated agent. However, the propagation of the endogenous murine PrPSc was favored, allowing us to speculate that host-specific factors may disturb the interference caused by the coexpression of an exogenous second PrPC. IMPORTANCE: Prion propagation can be interfered with by the expression of a second prion protein in the host. In the present study, we investigated prion propagation in a host expressing two different prion protein genes. Our findings indicate that the ability of the second prion protein to interfere with prion propagation is related to the transmissibility of the prion in the host expressing only the interfering prion protein. The interference detected occurs in a prion strain-dependent manner. Interestingly, a bias favoring the propagation of the murine PrP allele has been observed. These results open the door to future studies in order to determine the role of host factors other than the PrP amino acid sequence in the interference in prion propagation.