In the present study we used the water-soluble short chain phosphatidylcholine analogue L-alpha-dibutyryl-glycero-3-phosphatidylcholine (diC4PC) to investigate the mechanism involved in the canalicular secretion of phospholipids in rat liver. Uptake of 14C-labeled di-C4PC was studied in isolated microsomes as well as in basolateral (sinusoidal) and canalicular plasma membrane vesicles. Saturable uptake of diC4PC into an osmotically active space was observed in microsomes and canalicular membrane vesicles. In contrast, diC4PC uptake into basolateral membrane vesicles could be accounted for by cross-contamination with endoplasmic reticulum and canalicular membrane vesicles. Whereas the Km values for diC4PC uptake (37 degrees C) were similar in microsomes (7.4 +/- 2.6 mM) and canalicular membrane vesicles (8.2 +/- 2.0 mM), the Vmax values were approximately 2-fold higher in canalicular membrane vesicles (29.6 +/- 2.7 nmol/mg of protein x min) than in microsomes (16.7 +/- 2.1 nmol/mg of protein x min). Furthermore, Pronase treatment of the membrane vesicles reduced diC4PC uptake by 34-54% in both subfractions, whereas the D-[14C]glucose-accessible water space was only reduced by approximately 20%. These data provide direct evidence for the presence of a protein-mediated phosphatidylcholine translocating activity in the canalicular membrane of rat hepatocytes. This canalicular "flippase" has kinetic properties similar to those described previously in microsomes and provides a potential pathway for the translocation of bile salt dissolvable biliary phospholipids to the exoplasmic leaflet of the canalicular membrane.