ABSTRACT:In the present study, we attempted to identify the membrane permeation process(es) primarily involved in the molecular-weightdependent biliary excretion of ␤-lactam antibiotics. A search of the literature indicated that the molecular weight threshold operates mainly in the transport process across bile canalicular membranes. We confirmed that biliary clearance of the model biliaryexcretion-type cephalosporin cefoperazone was reduced to 10% of the control in Eisai hyperbilirubinemic rats, which are genetically deficient in multidrug resistance-associated protein (Mrp) 2, indicating that Mrp2 plays a major role as an efflux transporter on the canalicular membranes. ATP-dependent uptake of several cephalosporins including cefoperazone, cefbuperazone, cefpiramide, and ceftriaxone, all of which are mainly excreted into bile, was confirmed in membrane vesicles from Sf9 cells transfected with rat Mrp2. Both the inhibitory potency of the cephalosporins for Mrp2-mediated transport and the uptake of cephalosporins by Mrp2-expressing vesicles were molecular weight-dependent, suggesting that Mrp2 is one of the major transporters involved in molecular weight-dependent biliary excretion. An uptake study in membrane vesicles of Sf9 cells transfected with breast cancer resistance protein (Bcrp) revealed that Bcrp accepts cefoperazone, cefbuperazone, cefpiramide, cefotetan, ceftriaxone, cefotiam, cefamandole, and cefazolin as substrates, and Bcrp-mediated transport was also molecular weight-dependent, suggesting that Bcrp also contributes to molecular weight-dependent biliary excretion of ␤-lactam antibiotics in rats.