The antibiotics, rifamycin SV and rifampicin, are known to interfere with hepatic bile salt and organic anion uptake. The aim of this study was to explore which transport systems are affected. In short-term-cultured rat hepatocytes, low concentrations (10 micromol/L) of both compounds inhibited mainly sodium-independent taurocholate uptake, whereas higher concentrations (100 micromol/L) also inhibited sodium-dependent taurocholate uptake. In Xenopus laevis oocytes expressing the Na(+)/taurocholate cotransporting polypeptide (Ntcp), high rifamycin SV and rifampicin concentrations were required for inhibition of taurocholate uptake. In contrast, sodium-independent taurocholate uptake mediated by the organic anion transporting polypeptides, Oatp1 and Oatp2, was already substantially inhibited by 10 micromol/L rifamycin SV. Rifampicin potently inhibited Oatp2-mediated taurocholate uptake, but did not interfere with Oatp1-mediated taurocholate uptake. Similar effects of rifamycin SV and rifampicin were found for Oatp1- and Oatp2-mediated estradiol-17beta-glucuronide transport. Dixon plot analysis yielded a pattern compatible with competitive inhibition of estradiol-17beta-glucuronide transport with K(i) estimates of 6.6 micromol/L and 7.3 micromol/L for rifamycin SV-induced inhibition of Oatp1 and Oatp2, respectively, and of 1.4 micromol/L for rifampicin-induced inhibition of Oatp2. These results demonstrate that rifamycin SV and rifampicin exhibit differential inhibition on Oatp1 and Oatp2, and identify rifampicin as a selective Oatp2 inhibitor. The data indicate that these inhibitors can be used to determine the in vivo relevance of Oatp1 and Oatp2 for the overall bioavailability and disposition of drugs and other Oatp1/2 substrates.