About 95% of the bile phospholipids are phosphatidylcholine. Although the fractions of phosphatidylcholine and of both aminophospholipids phosphatidylserine and phosphatidylethanolamine in the canalicular membrane are in the same order of about 35% of total lipids, both aminophospholipids are almost absent from the bile. To rationalize this observation, we studied the intracellular uptake of various fluorescent phospholipid analogues and their subsequent enrichment in the bile canaliculus (BC) of HepG2 cells. Diacylaminophospholipid analogues but not phosphatidylcholine analogues became rapidly internalized by an aminophospholipid translocase (APLT) activity in the plasma membrane of HepG2 cells. We observed only low labeling of BC by diacylaminophospholipids but extensive staining by phosphatidylcholine analogues. In the presence of suramin, known to inhibit APLT, a strong labeling of BC by diacylaminophospholipid analogues was found that declined to a level observed for control cells after removal of suramin. Unlike diacylphosphatidylserine, diether phosphatidylserine analogue, which is not an appropriate substrate of APLT, accumulated in the BC. The correlation between low labeling of BC and an APLT-mediated transbilayer movement suggests the presence of an APLT activity in the canalicular membrane that prevents exposure of aminophospholipids to the bile.