Purpose: Due to their broad substrate specifity and their expression in a variety of tissues the H+/peptide cotransporters PEPT1 und PEPT2 play an important role in absorption and distribution of therapeutic peptidomimetics. The aim of this work was the functional characterization of transport of peptidomimetics by PEPT1 and PEPT2 at the epithelia of intestine, kidney and extrahepatic biliary duct. Such investigations on drug-transporting carriers can provide important information regarding the optimization of drug delivery. Methods: The studies were performed in vitro by use of epithelial cell lines. Jejunal transport of substances was investigated by means of the human colon carcinoma cell line Caco-2, which expresses PEPT1. For the investigation of substrate transport by PEPT2 at the proximal renal tubulus the SKPT cell line was used; for the characterization of transport of substances at PEPT1 of the extrahepatic bile duct the human carcinoma cell line SK-ChA-1 was employed. Main Results: Delta-aminolevulinic acid displays a medium affinity for PEPT1 and is transported by this carrier expressed in epithelial extrahepatic bile duct cells. The cell line SK-ChA-1 represents a convenient model for studying drug delivery at this tissue. The dipetidomimetic alafosfalin displays a high affinity to the intestinal PEPT1 and the renal PEPT2, respectively. It is actively transported by these carriers as well. Compared to the dipeptide alanylalanin, in alafosfalin the C-terminal carboxyl group has been replaced by phosphonate. The thiazolidids among the aminoacyl compounds investigated displayed a higher affinity to the intestinal PEPT1 than their pyrrolidide analogues. Hydrophilic amino acid residues reduced affinity as well. Moreover, an additional cyano group at the second carbon atom of the pyrrolidine ring of the isoleucyl compound strongly augmented affinity to the intestinal PEPT1. In a further study various therapeutic peptidomimetics were screened regarding their affinity to the intestinal PEPT1. So, for bestatin and other compounds a medium or low affinity was determined, respectively.