The oligopeptide transporter PEPT1, which is located in the intestinal brush border membrane, provides a major mechanism for protein absorption in the human intestine. Considering not only the nutritional importance of PEPT1, but also its pharmacological relevance for oral availability of peptidomimetics, structural information about substrate-protein interaction are highly relevant for future rational design of orally active peptide mimetics. We investigate the structural requirements for substrates, mainly of dipeptide, dipeptide- and amino acid derivatives, to be accepted by the peptide transport system. This transporter has been extensively studied with the human intestinal cell line Caco-2, who expressing the transportprotein spontaneously. Several amino acid amides were able to inhibit the uptake of [14C]glycylsarcosine in Caco-2 cells like natural peptides. We conclude that the intestinal H+/peptide cotransport system PEPT1 accepts different amino acid aryl amides as substrates. The affinity of dipeptide- and dipetide derivatives is dependent on a free α-amino-group and a free C-terminal carboxylic group, on stereospecificity and a hydrophobic side chain protecting group. Furthermore we have shown that a peptide bond is not an essential structural requirement.