L-Proline, D-proline, GABA, taurine, L-alanine and many other related compounds are transported into Caco-2 cells by the H⁺coupled amino acid transporter PAT1, cloned recently from brain and intestine. Transport occurs uphill and depends strongly on an inward directed H⁺-gradient. The carrier accepts many therapeutically active proline derivatives as substrates. We found that L-tryptophan and its derivatives tryptamine, 5-hydroxy-L-tryptophan, serotonin and indole-3-propionic acid function as naturally occurring PAT1 inhibitors which are recognized with affinitiy constants similar to those of prototype substrates but are not transported by the carrier. In preliminary RT-PCR experiments, the mRNA of both PAT1 and the recently cloned Na⁺-dependent imino acid transporter 1 (SIT1) was found in Caco-2 cells. We also investigated the influence of the H⁺-gradient on the kinetic parameters of L-proline transport in Caco-2 cells by measuring L-[³H]proline uptake in the absence/presence of a H⁺gradient and the histidyl residue blocking agent diethylpyrocarbonate (DEPC). Both extracellular protons and DEPC affected only the affinity constants of drug transport via PAT1 with no effect on maximal transport velocity.