Several proteins linked to neurodegenerative diseases, such as the beta-amyloid precursor protein, amyloid beta-peptide, beta-secretase, and tau, undergo selective polarized sorting. We investigated polarized sorting of the mammalian prion protein (PrP(C)) and its homologue doppel (Dpl). In contrast to Dpl, which accumulates on the apical surface, PrP(C) is targeted selectively to the basolateral side in Madin-Darby canine kidney cells. An extensive deletion and domain swapping analysis revealed that the internal hydrophobic domain (HD) of PrP (amino acids 113-133) confers basolateral sorting in a dominant manner. PrP mutants lacking the HD are sorted apically, while Dpl chimeras containing the HD of PrP are directed to the basolateral membrane. Furthermore, a pathogenic PrP missense mutation within the HD leads to aberrant apical sorting of PrP as well.