The mammalian proton-dependent peptide transporters PEPT1 and PEPT2 are mainly expressed in epithelial cells of small intestine and kidney and share a broad substrate specificity ranging from di- and tripeptides to peptidomimetics like b-lactam antibiotics and ACE inhibitors. We have identified the E. coli ydgR gene showing homology to PEPT1 and PEPT2, have cloned it and overexpressed the protein. The YdgR protein was characterized in vivo by transport assays using the fluorescent dipeptide b-Ala-Lys-Amca. Transport was not markedly altered in the absence of sodium, but completely abolished in the presence of 10 mM CCCP indicating a proton-dependent transport mechanism. The YdgR protein appears to be specific for di- and tripeptides and the substrate recognition pattern was very similar to the mammalian peptide transporter PEPT1. Even peptidomimetics like b-lactam antibiotics display good substrate affinities for interaction with YdgR. Large quantities of YdgR were purified to homogeneity and transmission electron microscopy of detergent-solubilized protein suggested YdgR to exist as a monomer displayed by round-shaped particles with a diameter of ~8 nm. From these data, we conclude that the YdgR protein is an excellent model to study protein-substrate interactions as well as the molecular structure of a peptide transporter.