The peptide transporter PEPT2 is a polytopic transmembrane protein that mediates the cellular uptake of di- and tripeptides and a variety of peptidomimetics. It is widely expressed in mammalian tissues including kidney, lung, mammary gland choroids plexus and glia cells. In renal tubular cells PEPT2 is exclusively found at the apical membrane. The molecular mechanisms underlying this polarized expression and targeting to the brush border membrane are not known. We have explored the role of the 36 carboxyterminal amino acid residues in PEPT2 trafficking and apical expression. EGFP-tagged PEPT2 wildtype transporter and various truncated and mutant proteins were expressed in the polarized proximal tubule cell lines SKPT and OK and the cellular distribution of the fusion proteins was assessed by confocal microscopy. Whereas deletion of the last 7 amino acids (delC7) did not alter PEPT2 surface expression, deletion of the next residue (delC8) or up to 30 terminal amino acids resulted in an impaired apical expression and distinct accumulation of mutant proteins in endosomal and lysosomal vesicles. Truncation of further amino acids (delC36) containing tyrosine-based motifs led to a rather diffuse intracellular distribution pattern. Mutations introduced at isoleucine⁷²⁰ (I720A) and leucine⁷²² (I722A) also caused impaired surface appearance. Internalization assays revealed a higher endocytotic rate of PEPT2 mutants I720A, L722A and delC36. Our data suggest that a three amino acid stretch (INL) and tyrosine-based motifs within the COOH-tail of PEPT2 are involved in its apical membrane localization and membrane steady state level.