Prions are infectious β-sheet-rich protein aggregates composed of misfolded prion protein (PrP^Sc) that do not possess coding nucleic acid. Prions replicate by recruiting and converting normal cellular PrP^C into infectious isoforms. In the same host species, prion strains target distinct brain regions and cause different disease phenotypes. Prion strains are associated with biophysically distinct PrP^Sc conformers, suggesting that strain properties are enciphered within alternative PrP^Sc quaternary structures. So far it is unknown how prion strains target specific cells and initiate productive infections. Deeper mechanistic insight into the prion life cycle came from cell lines permissive to a range of different prion strains. Still, it is unknown why certain cell lines are refractory to infection by one strain but permissive to another. While pharmacologic and genetic manipulations revealed subcellular compartments involved in prion replication, little is known about strain-specific requirements for endocytic trafficking pathways. This review summarizes our knowledge on how prions replicate within their target cells and on strain-specific differences in prion cell biology.