A phospholipid translocating protein from rat liver has been expressed in Xenopus laevis oocytes. Injection of oocytes with total rat liver messenger RNA (mRNA) resulted in the function expression of saturable uptake of the water soluble phophatidylcholine derivative L-alpha-dibutyroylglycero-3-phophatidylcholine (diC4PC), Kinetic studies revealed an apparent Km value of approximately 10 mmol/L, which is similar to the value previously obtained in isolated rat liver canalicular plasma membrane vesicles for an adenosine triphosphate (ATP)-independent phosphatidylcholine translocator. Size fractionation of total rat liver mRNA yielded an active mRNA species between 1.8 and 2.6 kb, that stimulated the expressed phophatidylcholine uptake activity approximately fivefold as compared with differently sized mRNA subfractions. This active mRNA size class is too small to code for the mdr2 P-glycoprotein, which has been suggested to function as an ATP-dependent canalicular phosphatidylcholine translocator. Hence, the data indicate that there are at least two separate polypeptides involved in phospholipid translocation from hepatocytes into bile.