While neuropathological features that define prion strains include spongiform degeneration and deposition patterns of PrP(Sc), the underlying mechanism for the strain-specific differences in PrP(Sc) targeting is not known. To investigate prion strain targeting, we inoculated hamsters in the sciatic nerve with either the hyper (HY) or drowsy (DY) strain of the transmissible mink encephalopathy (TME) agent. Both TME strains were initially retrogradely transported in the central nervous system (CNS) exclusively by four descending motor tracts. The locations of HY and DY PrP(Sc) deposition were identical throughout the majority of the incubation period. However, differences in PrP(Sc) deposition between these strains were observed upon development of clinical disease. The differences observed were unlikely to be due to strain-specific neuronal tropism, since comparison of PrP(Sc) deposition patterns by different routes of infection indicated that all brain areas were susceptible to prion infection by both TME strains. These findings suggest that prion transport and differential susceptibility to prion infection are not solely responsible for prion strain targeting. The data suggest that differences in PrP(Sc) distribution between strains during clinical disease are due to differences in the length of time that PrP(Sc) has to spread in the CNS before the host succumbs to disease.