Mitoxantrone is expelled by the ABCG2 multidrug transporter directly from the plasma membrane

►Hydrophobic drugs, gaining fluorescence in lipids, are expelled by GFP-tagged ABCG2. ►GFP-tagged ABCG2 allows tracking down the position of the plasma membrane. ►MX concentration can be measured separately in the plasma membrane and cell interior. ►Transport kinetic models disprove drug extrusion from the cytoplasm. ►These models support that MX transported by ABCG2 directly from the plasma membrane.