A complementary DNA (cDNA) encoding the rat H+/peptide cotransporter (PepT1) was isolated, and the transport characteristics of orally active beta-lactam antibiotics were assessed by measuring uptake into Xenopus oocytes expressing the rat PepT1. The rat PepT1 cDNA encoded a 710-amino acid protein with 77% identity to the rabbit PepT1. The message for rat PepT1 was approximately 2.9 kilobases and was found predominantly in the small intestine, whereas reverse transcription-polymerase chain reaction amplification revealed that the message was expressed both in the small intestine and in the kidney cortex. The 75-kDa protein was identified by translation of in vitro synthesized transcript of rat PepT1 cDNA by use of rabbit reticulocyte lysates and by Western blot analysis with a specific antibody against the rat PepT1. When expressed in Xenopus oocytes, rat PepT1 stimulated the uptake of ceftibuten (anion) and cephradine (zwitterion) in the presence of an inward H+ gradient, and the expressed uptake was inhibited by excess dipeptides. Kinetic analysis revealed that ceftibuten has 14-fold higher affinity for the rat PepT1 than cephradine. These findings suggest that the rat PepT1 mediates H(+)-coupled uphill transport of the oral beta-lactam antibiotics across the brush-border membranes of intestinal and renal proximal tubular cells.