This study was initiated to develop inhibitors of the intestinal H(+)/peptide symporter. We provide evidence that the dipeptide derivative Lys[Z(NO(2))]-Pro is an effective competitive inhibitor of mammalian PEPT1 with an apparent binding affinity of 5-10 microM. Characterization of the interaction of Lys[Z(NO(2))]-Pro with the substrate binding domain of PEPT1 has been performed in (a) monolayer cultures of human Caco-2 cells expressing PEPT1, (b) transgenic Pichia pastoris cells expressing PEPT1, and (c) Xenopus laevis oocytes expressing PEPT1. By competitive uptake studies with radiolabeled dipeptides, HPLC analysis of Lys[Z(NO(2))]-Pro in cells, and electrophysiological techniques, we unequivocally show that Lys[Z(NO(2))]-Pro binds with high affinity to PEPT1, competes competitively with various dipeptides for uptake into cells, but is not transported itself. Lack of transport was substantiated by the absence of Lys[Z(NO(2))]-Pro in Caco-2 cell extracts as determined by HPLC analysis, and by the absence of any positive inward currents in oocytes when exposed to the inhibitor. The fact that Lys[Z(NO(2))]-Pro can bind to PEPT1 from the extracellular as well as the intracellular site was shown in the oocyte expression system by a strong inhibition of dipeptide-induced currents under voltage clamp conditions. Our findings serve as a starting point for the identification of the substrate binding domain in the PEPT1 protein as well as for studies on the physiological and pharmacological role of PEPT1.