Background & Aims Hartnup amino acid transporter B0 AT1 (SLC6A19) is the major luminal sodium-dependent neutral amino acid transporter of small intestine and kidney proximal tubule. The expression of B0 AT1 in kidney was recently shown to depend on its association with collectrin (Tmem27), a protein homologous to the membrane-anchoring domain of angiotensin-converting enzyme (ACE) 2. Methods Because collectrin is almost absent from small intestine, we tested the hypothesis that it is ACE2 that interacts with B0 AT1 in enterocytes. Furthermore, because B0 AT1 expression depends on an associated protein, we tested the hypothesis that Hartnup-causing B0 AT1 mutations differentially impact on B0 AT1 interaction with intestinal and kidney accessory proteins. Results Immunofluorescence, coimmunoprecipitation, and functional experiments using wild-type and ace2 -null mice showed that expression of B0 AT1 in small intestine critically depends on ACE2. Coexpressing new and previously identified Hartnup disorder–causing missense mutations of B0 AT1 with either collectrin or ACE2 in Xenopus laevis oocytes showed that the high-frequency D173N and the newly identified P265L mutant B0 AT1 transporters can still be activated by ACE2 but not collectrin coexpression. In contrast, the human A69T and R240Q B0 AT1 mutants cannot be activated by either of the associated proteins, although they function as wild-type B0 AT1 when expressed alone. Conclusions We thus show that ACE2 is necessary for the expression of the Hartnup transporter in intestine and suggest that the differential functional association of mutant B0 AT1 transporters with ACE2 and collectrin in intestine and kidney, respectively, participates in the phenotypic heterogeneity of human Hartnup disorder.