The cellular prion protein (PrP(C)) was recently observed to co-purify with members of the LIV-1 subfamily of ZIP zinc transporters (LZTs), precipitating the surprising discovery that the prion gene family descended from an ancestral LZT gene. Here, we compared the subcellular distribution and biophysical characteristics of LZTs and their PrP-like ectodomains. When expressed in neuroblastoma cells, the ZIP5 member of the LZT subfamily was observed to be largely directed to the same subcellular locations as PrP(C) and both proteins were seen to be endocytosed through vesicles decorated with the Rab5 marker protein. When recombinantly expressed, the PrP-like domain of ZIP5 could be obtained with yields and levels of purity sufficient for structural analyses but it tended to aggregate, thereby precluding attempts to study its structure. These obstacles were overcome by moving to a mammalian cell expression system. The subsequent biophysical characterization of a homogeneous preparation of the ZIP5 PrP-like ectodomain shows that this protein acquires a dimeric, largely globular fold with an α-helical content similar to that of mammalian PrP(C). The use of a mammalian cell expression system also allowed for the expression and purification of stable preparations of Takifugu rubripes PrP-1, thereby overcoming a key hindrance to high-resolution work on a fish PrP(C).