Phosphatidylcholine (PC) translocation was studied in rat liver canalicular plasma membrane vesicles using a fluorescent PC analogue that permitted the quantitation of asymmetric PC distribution in the outer and inner leaflet of the vesicles. PC translocation to the outer leaflet of the canalicular membrane was stimulated by ATP and an ATP-regenerating system in a time and temperature-dependent manner resulting in 200 pmol PC translocated/mg protein per 30 min. A non-hydrolyzable ATP analogue did not support translocation. Translocating activity was observed with PC but not with phosphatidylethanolamine and was specific for inside-out oriented canalicular membrane vesicles. Addition of taurocholate (10 microM), a micelle-forming bile acid, enhanced ATP-dependent PC translocation 1.5 +/- 0.1-fold, whereas addition of taurodehydrocholate (10 microM), a non-micelle-forming bile acid, did not. These results indicate the presence of an ATP-dependent transporter that "flips" phosphatidylcholine from the inner to the outer leaflet of the rat bile canalicular plasma membrane from where it can become associated with bile acids in the canalicular lumen, thereby enhancing ATP-dependent flipping activity. Several lines of evidence suggest that the transporter is Mdr2 P-glycoprotein.