Many small oligopeptides are rapidly excreted unchanged into bile, which requires vectorial transport across the hepatocyte. To characterize the involved carrier system(s) at the canalicular membrane, studies were undertaken with vesicle preparations from the rat and the model pseudohexapeptide ditekiren. The initial uptake rate into inside-out-oriented vesicles was found to be ATP- and temperature-dependent and saturable. Kinetic analysis indicated the involvement of three processes: (1) an ATP-dependent carrier-mediated process (Km = 19.1 +/- 4.26 microM; mean +/- S.E.M.), Vmax = 140 +/- 29.4 pmol/mg of protein/15 sec), (2) an ATP-independent carrier-mediated transporter (Km = 17.2 +/- 9.58 microM, Vmax = 62.9 +/- 24.5 pmol/mg of protein/15 sec) and (3) a nonsaturable component. ATP-dependent uptake was inhibited by several other oligopeptides, which in the case of EMD 51921 was competitive. Cis-inhibition studies with known substrates for the canalicular bile salt (taurocholate), multispecific organic anion (glutathione disulfide) and P-glycoprotein (daunomycin, nicardipine, cyclosporin A) transporters indicated a major role for the latter carrier system. Inhibition of the initial uptake rate of ditekiren by daunomycin was found to be competitive in nature (Ki = 16 microM). These findings indicate that the biliary excretion of ditekiren and possibly other hydrophobic oligopeptides is mediated, in part, by P-glycoprotein and suggest a possible physiological role for this hepatic transporter.