The uptake system for beta-lactam antibiotics in the rabbit small intestine was investigated using brush-border membrane vesicles. After treatment of membrane vesicles with the reagent diethylpyrocarbonate (DEP), the uptake of orally active beta-lactam antibiotics with an alpha-amino group in the substituent at position 6 or 7 of the penam or cephem nucleus was significantly inhibited, whereas DEP-treatment had no inhibitory effect on the uptake of beta-lactam antibiotics without an alpha-amino group. The kinetic analysis revealed an apparent competitive inhibition indicating a decreased affinity of the transport system for alpha-amino-beta-lactam antibiotics. Substrates of the intestinal dipeptide transport system - dipeptides and alpha-amino-beta-lactam antibiotics - could protect the transport system from irreversible inhibition by DEP, whereas beta-lactam antibiotics without an alpha-amino group as well as amino acids or bile acids had no effect. Incubation of DEP-treated vesicles with hydroxylamine led to a partial restoration of the transport activity indicating that DEP may have led to a modification of a histidine residue of the transport protein. From the data presented we conclude that a specific interaction of the alpha-amino group in the substituent at position 6 or 7 of the penam or cephem nucleus presumably with a histidine residue of the transport protein is involved in the translocation process of orally active alpha-amino-beta-lactam antibiotics across the intestinal brush-border membrane.