1. The stereoselective transport of beta-lactam antibiotics has been investigated in the human intestinal epithelial cell line, Caco-2, by use of D-and L-enantiomers of cephalexin and loracarbef as substrates. 2. The L-isomers of cephalexin, loracarbef and dipeptides displayed a higher affinity for the oligopeptide/H (+)-symporter in Caco-2 cells than the D-isomers. This was demonstrated by inhibition of the influx of the beta-lactam,[3H]-cefadroxil. 3. By measurement of the substrate-induced intracellular acidification in Caco-2 cells loaded with the pH-sensitive fluorescent dye BCECF (2', 7'-bis (2-carboxyethyl)-5-(6)-carboxy-fluorescein), it was demonstrated for the first time that L-isomers of beta-lactams not only bind to the peptide transporter with high affinity but are indeed transported. 4. Efficient proton-coupled transport of L-beta-lactam antibiotics was also shown to occur in Xenopus laevis oocytes expressing the cloned peptide transporter PepT1 from rabbit small intestine. 5. Both cell systems therefore express a stereoselective transport pathway for beta-lactam antibiotics with very similar characteristics and may prove useful for screening rapidly the oral availability of peptide-derived drugs.