By utilizing the everted jejunum of rats, the initial uptake rates of several antibiotics were measured over a wide range of concentrations. The uptakes followed mixed-type kinetics involving saturable and non-saturable processes in parallel. The pertinent kinetic parameters for the uptake of each antibiotic were determined. The effect of cephalexin on the uptake of cyclacillin obeyed competitive inhibition kinetics, and the inhibition constant Ki was found to be equal to the Michaelis constant Kt for the uptake of cephalexin itself. In a similar way, the uptake of cephalexin was inhibited by cyclacillin. Uptakes of both cyclacillin and cephalexin were reduced significantly by several metabolic inhibitors. From the effect of temperature on the uptakes of cyclacillin and cephalexin, activation energies of 24.8 and 23.1 kcal/mole were obtained respectively. These results indicate the involvement of an active transport mechanism for cyclacillin and cephalexin. It was found that several dipeptides markedly inhibited the uptakes of cyclacillin and cefadroxil. Furthermore, the uptake of glycylglycine, a typical dipeptide, was inhibited by cyclacillin, cefadroxil, cephalexin, and cephradine. The kinetics of mutual inhibition of the uptakes of cyclacillin and glycylglycine were consistent with competitive-type inhibition. This is the first report which establishes, from a kinetic point of view, the involvement of a common transport system in the in vitro uptakes of the dipeptides and the antibiotics.