Brush border membrane vesicles from rabbit small intestine were used to study the intestinal uptake system for beta-lactam antibiotics. Benzylpenicillin inhibited the H+-dependent uptake of alpha-aminocephalosporins in a concentration-dependent manner suggesting a common transport system for alpha-aminocephalosporins and benzylpenicillin. Benzylpenicillin is therefore a suitable probe to characterize this transport system. Irradiation of [3H]benzylpenicillin using light sources having their maximum of radiation at 300 or 254 nm resulted in a covalent incorporation of radioactivity into penicillin binding proteins as was shown with serum albumin. Hence [3H]benzylpenicillin can be used for direct photoaffinity labeling of penicillin binding proteins in different cells and tissues. In brush border membrane vesicles from rabbit small intestine predominantly a membrane polypeptide with an apparent molecular weight of 127,000 was labeled by [3H]benzylpenicillin. Competition labeling experiments demonstrated that beta-lactam antibiotics--penicillins and cephalosporins--specifically interact with this protein, whereas amino acids, sugars or bile acids had no effect on the labeling pattern. Compounds which decreased the labeling of the 127,000 molecular weight membrane polypeptide also inhibited the H+-dependent uptake of the alpha-aminocephalosporin cephalexin into intestinal brush border membrane vesicles. These results suggest that a polypeptide of molecular weight 127,000 in the brush border membrane from rabbit small intestine is a constituent of a common transport system responsible for the uptake of orally effective beta-lactam antibiotics and dipeptides. beta-Lactam antibiotics which are not absorbed from the small intestine also bind from the luminal site to this transport system, but are not transported across the brush border membrane.