The bile salt export pump (BSEP) of hepatocyte secretes conjugated bile salts across the canalicular membrane in an ATP-dependent manner. The biliary bile salts of human differ from those of rat in containing a greater proportion of glycine conjugates and taurolithocholate 3-sulfate (TLC-S). In the present study, the transport properties of hBSEP and rBsep were investigated using membrane vesicles from HEK293 cells infected with recombinant adenoviruses containing hBSEP or rBsep cDNA. ATP-dependent uptake of radiolabeled glycine-, taurine-conjugated bile salts, and [(3)H]cholate was observed when hBSEP or rBsep was expressed. Comparison of initial uptake rates indicated that for both transporters, taurine-conjugated bile salts were transported more rapidly than glycine-conjugated bile salts, however, hBSEP transported glycine conjugates to an extent that was approximately 2-fold greater than rBsep. In addition, [(3)H]TLC-S was significantly transported by hBSEP, and hardly transported by rBsep. The mean K(m) value for the uptake of [(3)H]TLC-S by hBSEP was 9.5+/-1.5 microM, a value similar to that for hMRP2 (8.2+/-1.3 microM). In conclusion, both hBSEP and rBsep transport taurine-conjugated bile salts better than glycine-conjugated bile salts, but hBSEP transports glycine conjugates to a greater extent as compared to rBsep. TLC-S, which is present in human bile but not rodent bile, is more avidly transported by hBSEP compared with rBsep.