Abstract The interaction of two renin inhibitors, S 86 2033 and S 86 3390, with the uptake system for β-lactam antibiotics and small peptides in the brush border membrane of enterocytes from rabbit small intestine was investigated using brush border membrane vesicles. Both renin inhibitors inhibited the uptake of the orally active cephalosporin cephalexin into brush border membrane vesicles from rabbit small intestine in a concentration-dependent manner. 1.1 mM of S 86 3390 and 2.5 mM of S 86 2033 led to a half-maximal inhibition of the H +-dependent uptake of cephalexin. Both renin inhibitors were stable against peptidases of the brush border membrane. The uptake of cephalexin into brush border membrane vesicles (1 min of incubation) was competitively inhibited by S 86 2033 and S 86 3390 suggesting a direct interaction of these compounds with the intestinal peptide uptake system. The renin inhibitors are transported across the brush border membrane into the intravesicular space as was shown by equilibrium uptake studies dependent upon the medium osmolarity. The uptake of s 86 3390 was stimulated by an inwardly directed H +-gradient and occurred with a transient accumulation against a concentration gradient (overshoot phenomenon). The renin inhibitors S 86 2033 and 86 3390 also caused a concentration-dependent inhibition in the extent of photoaffinity labeling of the putative peptide transport protein of apparent M r 127 000 in the brush border membrane of small intestinal enterocytes. In conclusion, these studies show that renin inhibitors specifically interact with the intestinal uptake system shared by small peptides and β-lactam antibiotics.