The present study was designed to identify the organic anion transporting polypeptide (OATP) molecule(s) responsible for the uptake of beta-lactam antibiotics in human liver, using cryopreserved hepatocytes, as well as Xenopus oocytes and cultured cells expressing human OATPs. Nafcillin uptake by human hepatocytes was saturable with a Km of 533 microM. In vitro uptake studies revealed that OATP1B3 and OATP1B1 transported nafcillin with Km values of 74 microM and 11 mM, respectively. Analysis by the relative activity factor method suggested that OATP1B3 contributes mainly to nafcillin uptake and OATP1B1 contributes moderately. This conclusion was supported by the results of a study with selective inhibitors. Furthermore, OATP1B3 transported six other beta-lactam antibiotics, and their uptake clearances by OATP1B3 correlated well with those mediated by rat Oatp1a4, which is the predominant contributor to basolateral uptake of nafcillin by rat hepatocytes. These findings suggest that OATP1B3 plays a major role in the hepatic uptake of beta-lactam antibiotics in humans, and probably corresponds functionally to Oatp1a4 in rat liver.