The human MDR1 P-glycoprotein (Pgp) extrudes a variety of drugs across the plasma membrane. The homologous MDR3 Pgp is required for phosphatidylcholine secretion into bile. After stable transfection of epithelial LLC-PK1 cells, MDR1 and MDR3 Pgp were localized in the apical membrane. At 15 degrees C, newly synthesized short-chain analogs of various membrane lipids were recovered in the apical albumin-containing medium of MDR1 cells but not control cells. MDR inhibitors and energy depletion reduced apical release. MDR3 cells exclusively released a short-chain phosphatidylcholine. Since no vesicular secretion occurs at 15 degrees C, the short-chain lipids must have been translocated by the Pgps across the plasma membrane before extraction into the medium by the lipid-acceptor albumin.