Lactococcus lactis possesses an ATP-dependent drug extrusion system which shares functional properties with the mammalian multidrug resistance (MDR) transporter P-glycoprotein. One of the intriguing aspects of both transporters is their ability to interact with a broad range of structurally unrelated amphiphilic compounds. It has been suggested that P-glycoprotein removes drugs directly from the membrane. Evidence is presented that this model is correct for the lactococcal multidrug transporter through studies of the extrusion mechanism of BCECF-AM and cationic diphenylhexatriene (DPH) derivatives from the membrane. The non-fluorescent probe BCECF-AM can be converted intracellularly into its fluorescent derivative, BCECF, by non-specific esterase activities. The development of fluorescence was decreased upon energization of the cells. These and kinetic studies showed that BCECF-AM is actively extruded from the membrane before it can be hydrolysed intracellularly. The increase in fluorescence intensity due to the distribution of TMA-DPH into the phospholipid bilayer is a biphasic process. This behaviour reflects the fast entry of TMA-DPH into the outer leaflet followed by a slower transbilayer movement to the inner leaflet of the membrane. The initial rate of TMA-DPH extrusion correlates with the amount of probe associated with the inner leaflet. Taken together, these results demonstrate that the lactococcal MDR transporter functions as a 'hydrophobic vacuum cleaner', expelling drugs from the inner leaflet of the lipid bilayer. Thus, the ability of amphiphilic substrates to partition in the inner leaflet of the membrane is a prerequisite for recognition by multidrug transporters.