Loracarbef, cefixime and cefuroxime axetil are beta-lactam antibiotics that are administered orally. Oral absorption of loracarbef is nearly complete, while that of cefixime and cefuroxime axetil is 30-50%. To investigate this we used the human intestinal cell line Caco-2 that possesses the proton-dependent peptide transporter that takes up cephalexin and cefaclor. Drug uptake was measured at pH 6 by high performance liquid chromatography or with radioactively labelled drug. The initial uptake rate of 1 mM cefixime was lower than that of 1 mM loracarbef. By 2 h both drugs were concentrated intracellularly against a gradient; however, the accumulation of cefixime was only 40% of that of loracarbef. The uptake rate of both drugs was sodium-independent, temperature- and energy-dependent, and was inhibited by dipeptides, cephalexin, cefaclor, but not by amino acids. Kinetic analysis of the concentration-dependence of the uptake rates for loracarbef and cefixime indicated that diffusion and a single transport system were responsible for uptake. The kinetic parameters for loracarbef and cefixime, respectively, were: Km values of 8 and 17 mM and Vmax values of 6.5 and 2 nmol/min per mg protein. Loracarbef and cefixime were competitive inhibitors of each other's uptake. By contrast, cefuroxime axetil was taken up and rapidly hydrolyzed to cefuroxime by Caco-2 cells. Cefuroxime axetil uptake was not dependent on energy and was not affected by dipeptides. Thus, cefuroxime axetil apparently enters Caco-2 cells by simple diffusion. By contrast, loracarbef and cefixime share a common transport mechanism, the proton-dependent dipeptide transporter. Cefixime was taken up less well than loracarbef due to a substantial reduction in the turnover rate and decreased affinity of the transporter for cefixime.