Prions are infectious pathogens essentially composed of PrP(Sc), an abnormally folded form of the host-encoded prion protein PrP(C). Constrained steric interactions between PrP(Sc) and PrP(C) are thought to provide prions with species specificity and to control cross-species transmission into other host populations, including humans. We compared the ability of brain and lymphoid tissues from ovine and human PrP transgenic mice to replicate foreign, inefficiently transmitted prions. Lymphoid tissue was consistently more permissive than the brain to prions such as those causing chronic wasting disease and bovine spongiform encephalopathy. Furthermore, when the transmission barrier was overcome through strain shifting in the brain, a distinct agent propagated in the spleen, which retained the ability to infect the original host. Thus, prion cross-species transmission efficacy can exhibit a marked tissue dependence.