Cefditoren is a third-generation cephalosporin developed by Meiji Seika Kaisha Ltd. Many beta-lactam antibiotics are transported by the H+/peptide symporters PEPT1 and PEPT2 that are preferentially expressed in the luminal membrane of the intestine and kidney, respectively. In this study, we employed everted small intestinal preparations, in situ jejunal perfusion, and Caco-2 cells as models to determine the effects of glycylsarcosine (Gly-Sar) and clonidine on uptake and transport of cefditoren. In vivo, rats were administered cefditoren (10 mg/kg) intravenously, in the absence and presence of Gly-Sar (10 mg/kg). The effects of Gly-Sar coadministration on biliary and urinary cefditoren excretion were investigated. Gly-sar significantly decreased cefditoren uptake and transport in the three in vitro and in situ models. A kinetic study showed that cefditoren transport by PEPT1 in Caco-2 cells had K(m) and V(max) values of 0.94+/-0.11 mM and 0.49+/-0.09 nmol/mg protein/5 min, respectively. Clonidine induced a 50% increase of cefditoren absorption across the intestinal mucosa after intravenous infusion, in the jejunal perfusion model. In vivo, biliary and urinary excretions over 6 h were an average of 34% and 4% of the administered cefditoren respectively. Gly-Sar coadministration increased the renal clearance of cefditoren by 200% and values of CL(urine) for cefditoren in the presence of 50 mM Gly-Sar were significantly higher than the controls. Biliary excretion was unchanged, however, compared to cefditoren alone. This study provides the first in vitro and in vivo evidence that cefditoren is a substrate of PEPT1.