By using isolated rat hepatocytes, the mechanism of uptake of a zwitterionic beta-lactam antibiotic, cephalexin, was clarified. The uptake followed the combination of saturable carrier-mediated and nonsaturable first-order rate processes. The kinetic parameters were estimated as follows (mean +/- SD): maximum uptake rate (Vmax), 2.28 +/- 0.24 nmol/min/mg of protein; Michaelis constant (Kt), 6.28 +/- 0.31 mM and first-order rate constant (kd), 0.11 +/- 0.012 nmol/min/mg of protein/mM. There was no inhibitory effect by amino acids, dipeptides or organic cations, whereas an organic anion, probenecid, markedly inhibited the hepatic uptake of cephalexin. Several beta-lactam antibiotics including zwitterionic and anionic derivatives inhibited cephalexin uptake significantly. The inhibition kinetics revealed that benzylpenicillin and the stereo-isomer l-cephalexin competitively inhibited cephalexin uptake. Furthermore, the efflux of cephalexin from the cells was stimulated by adding benzylpenicillin in the extracellular medium. These results demonstrated that all beta-lactam antibiotics have a common transport system with an organic anion such as probenecid, irrespective of their ionic charges, though a cationic charge on the molecule is less advantageous for being recognized by the carrier system.