The biliary excretion mechanism of three derivatives of BQ-123, an anionic cyclopentapeptide, was examined using isolated canalicular membrane vesicles (CMVs) from Sprague-Dawley rats. The uptake by CMV of BQ-485, a linear peptide, BQ-518, a cyclic peptide, and compound A, a cyclic peptide with a cationic moiety, was stimulated by ATP. An "overshoot" phenomenon and saturation were observed for the ATP-dependent uptake of these three peptides. The Michaelis-Menten constants (Km) for the uptake of BQ-485 and BQ-518 were comparable to the inhibition constants (Ki) for their inhibitory effects on ATP-dependent [3H]BQ-123 uptake. The uptake of BQ-485 showed the highest value and was inhibited by BQ-123 with a Ki that was comparable to the Km for BQ-123 uptake. The ATP-dependent uptake of BQ-123, BQ-485, and BQ-518 was much lower in CMVs from Eisai hyperbilirubinemic rats, a strain having a hereditary defect of the canalicular multispecific organic anion transporter (cMOAT). These results suggest that both BQ-485 and BQ-518 principally share the cMOAT transporter with BQ-123. Compound A almost completely inhibited BQ-123 uptake, although its ATP-dependent uptake was much lower than that of the other three peptides. The ATP-dependent uptake of compound A was not very different in Sprague-Dawley rats and Eisai hyperbilirubinemic rats and was not inhibited by S-(2, 4-dinitrophenyl)-glutathione, a typical substrate for cMOAT. Thus, although compound A inhibits cMOAT-mediated transport, its own transport by cMOAT is minimal and mediated by another transporter. This low degree of primary active transport by cMOAT may be the principal reason for its relatively longer residence in the circulation.