Several ABC proteins are expressed in the canalicular membrane. These include BSEP, involved in monoanionic bile acid (BA) secretion, MRP2, that exports drugs and conjugated bilirubin, and BCRP whose role in this location is not known. The aim of this study was investigate whether BCRP is involved in the biliary secretion of BA derivatives. The mRNA of the transporter Oatp1/1a1 plus that of an exporting pump (BCRP, Bsep or Mrp2) were microinjected into Xenopus laevis oocytes, and 48 h later, unconjugated (cholic) or conjugated (glycocholic, taurocholic, taurodeoxycholic, taurochenodeoxycholic, tauroursodeoxycholic) BAs, as well as the BA derivative cholylglycylamido-fluorescein (CGamF) were used as substrate in transport studies. The uptake of all these compounds was increased by expression of Oatp1/1a1. The co-expression with Bsep, but not with BCRP, reduced the cellular content of unconjugated and conjugated BAs. Co-expression with BCRP but not with Mrp2, reduced the cellular content of CGamF, which was prevented by Cyclosporin-A, an inhibitor of ABC proteins. The ability of BCRP to transport CGamF was verified using WIF-B9/R cells, which form bile canaliculi-like structures and that overexpress BCRP. These cells were able to take up and secrete CGamF into these structures. When the effect of verapamil, probenecid, taurocholate and Hoechst-33342 on CGamF transport by WIF-B9/R cells was investigated, the strongest inhibitory effect of CGamF efflux was induced by Hoechst-33342, a BCRP substrate. In conclusion, these results suggest that BCRP may play a role in the secretion into bile of BA derivatives.