A large number of structurally distinct electrophiles are conjugated to glutathione within hepatocytes, and the resulting glutathione S-conjugates are selectively transported across the canalicular membrane into bile. To test the hypothesis that a single multi-specific, ATP-dependent carrier mediates biliary secretion of glutathione S-conjugates, the present study compared the driving forces and substrate specificity for canalicular transport of S-ethylglutathione (ethyl-SG), a low molecular weight and relatively hydrophilic thioether, and S-(2,4-dinitrophenyl)-glutathione (DNP-SG), a larger and more hydrophobic anion, using isolated rat liver canalicular membrane vesicles. In agreement with previous findings, DNP-SG transport was stimulated by ATP, although there was considerable transport in the absence of ATP. ATP-independent DNP-SG transport was unaffected by a Na+ gradient, was enhanced by a valinomycin-induced K+ diffusion potential, and was saturable, with both high affinity (Km = 8 +/- 2 microM) and low affinity (Km = 0.5 +/- 0.1 mM) components. High affinity ATP-independent DNP-SG uptake was cis-inhibited by GSH, GSH monoethyl ester, glutathione S-conjugates, other gamma-glutamyl compounds, sulfobromophthalein, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). In contrast, ATP-dependent DNP-SG uptake was unaffected by GSH, GSH ester, S-methyl glutathione, or S-carbamidomethyl glutathione, but was strongly inhibited by sulfobromophthalein, DIDS, and by high molecular weight and relatively hydrophobic glutathione S-conjugates. Transport of the low molecular weight ethyl-SG conjugate was only minimally stimulated by ATP (10-20%). ATP-independent ethyl-SG uptake was electrogenic, saturable (Km = 10 +/- 1 microM) and was inhibited by GSH and all glutathione S-conjugates tested. These findings indicate the presence of multiple canalicular transport mechanisms for glutathione S-conjugates and demonstrate that the physicochemical properties of the S moiety are major determinants of transport. Relatively high molecular weight hydrophobic conjugates are substrates for both ATP-dependent and -independent mechanisms, whereas low molecular weight glutathione S-conjugates are transported largely by electrogenic carriers.