Plasma membranes in eukaryotic cells display asymmetric lipid distributions with aminophospholipids concentrated in the inner and sphingolipids in the outer leaflet. This asymmetry is maintained by ATP-driven lipid transporters whose identities are unknown. The yeast plasma membrane contains two P-type ATPases, Dnf1p and Dnf2p, with structural similarity to ATPase II, a candidate aminophospholipid translocase from bovine chromaffin granules. Loss of Dnf1p and Dnf2p virtually abolished ATP-dependent transport of NBD-labeled phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine from the outer to the inner plasma membrane leaflet, leaving transport of sphingolipid analogs unaffected. Labeling with trinitrobenzene sulfonic acid revealed that the amount of phosphatidylethanolamine exposed on the surface of Deltadnf1Deltadnf2 cells increased twofold relative to wild-type cells. Phosphatidylethanolamine exposure by Deltadnf1Deltadnf2 cells further increased upon removal of Drs2p, an ATPase II homolog in the yeast Golgi. These changes in lipid topology were accompanied by a cold-sensitive defect in the uptake of markers for bulk-phase and receptor-mediated endocytosis. Our findings demonstrate a requirement for Dnf1p and Dnf2p in lipid translocation across the yeast plasma membrane. Moreover, it appears that Dnf1p, Dnf2p and Drs2p each help regulate the transbilayer lipid arrangement in the plasma membrane, and that this regulation is critical for budding endocytic vesicles.