Complementary DNA clone encoding the rat small-intestinal oligopeptide transporter, rat PepT1, was isolated from a rat jejunal cDNA library by cross-hybridization with a rabbit PepT1 cDNA probe. The cDNA sequence indicated that rat PepT1 is composed of 710 amino acids and shows 77% and 83% amino acid sequence identity with rabbit and human PepT1, respectively. Hydropathy analysis reveals 12 putative transmembrane domains, with a long (204 amino acids) hydrophilic segment containing five potential N-glycosylation sites between transmembrane domains 9 and 10, in the predicted rat protein. Northern blot analysis detected rat PepTl mRNA in the small intestine and kidney. Complementary RNA (cRNA) of PepT1 was synthesized in vitro transcription and injected into Xenopus laevis oocytes to evaluate its transport function. Uptake of a dipeptide, [14C] glycylsarcosine by the oocytes was about 10-times higher than that by oocytes injected with water. The cRNA induced-uptake was enhanced in the presence of an inwardly directed proton gradient and was specifically inhibited by dipeptides and tripeptide, whereas their constitutive amino acids, tetra- and penta-peptide were not inhibitory. Accordingly, the isolated clone was confirmed as a rat intestinal proton-coupled oligopeptide transporter, PepT1. Zwitterionic and dianionic cephalosporins, cefadroxil and ceftibuten, were also specifically taken up by the oocytes injected with cRNA, Furthermore, mutual inhibitory effects on the uptakes were observed between glycylsarcosine and these cephalosporins. Hybrid depletion of the expression of rat or rabbit PepT1 in Xenojus laevis oocytes injected with intestinal total mRNA was studied using antisense oligonucleotides corresponding to the 5'-coding region of PepT1. In oocytes injected with both mRNA hybridized with each antisense oligonucleotide, ceftibuten uptakes were almost completely abolished. However, these uptake were not influenced with respective sense oligonucleotide. These results suggest that PepT1 functions for the intestinal absorption of oligopeptides and, β-lactam antibiotics.