The recently cloned proton-coupled amino acid transporter 1 (PAT1) is expressed at the brush border membrane of the human intestine. The system may serve as a new oral drug delivery route. In this study the transport mechanism of PAT1 regarding the cotransported proton was investigated using the human intestinal cellline Caco-2 and HRPE-PAT1-cells. H+ altered only the apparent affinity of L-proline transport and not the maximal transport velocity. Similarly, treatment of the cells with diethylpyrocarbonate (DEPC), known to chemically modify histidyl residues and block their function, affected only the Kt value of L-proline transport. H+ stimulated hPAT1 primarily by increasing the substrate affinity with no detectable influence on the maximal transport velocity of the transporter. Three histidine residues are conserved among the H+-coupled amino acid transporters PAT1 to 4 from different animal species. Histidine 55 was found to be essential for the catalytic activity of hPAT1. It might be responsible for binding and translocation of H+ in the course of cellular amino acid uptake by PAT1. The structural requirements for PAT1 substrates were systematically analyzed the by testing 87 amino acids, proline homologs, indoles, and derivatives. For aliphatic amino acids, a blocked carboxyl group, the distance between amino and carboxyl group, and the position of the hydroxyl group are affinity limiting factors. Methylation of the amino group enhances substrate affinity and hetero atoms in the proline template are well tolerated. Aromatic α-amino acids display low affinity. Serotonin, L-tryptophan and tryptamine bind to PAT1 with potencies similar to the prototype substrates, inhibit transport function but are not transported by this carrier protein. They may be considered as the carriers’ naturally occurring inhibitors that may alter the transport function of PAT1. The transport of known and new pharmaceutically active compounds via PAT1 was demonstrated. PAT1 is a promising candidate for new ways of oral drug delivery.