P-glycoprotein (P-gp) is a plasma membrane ATP-binding cassette transporter, responsible for multidrug resistance in tumor cells. P-gp catalyzes the ATP hydrolysis-dependent efflux of numerous amphiphilic compounds of unrelated chemical structures. In the absence of any identified substrate, P-gp exhibits an apparently futile, basal ATPase activity. By using native membrane vesicles containing high amounts of P-gp, we show here that (i) this basal ATPase activity is tightly dependent on the presence of cholesterol in the membrane; (ii) the stimulation of P-gp ATPase activity by drugs transported by P-gp is higher in the absence than in the presence of cholesterol and, conversely, the stimulation of P-gp ATPase activity by cholesterol is higher in the absence than in the presence of known P-gp substrates; (iii) P-gp mediates the ATP-dependent relocation of cholesterol from the cytosolic leaflet to the exoplasmic leaflet of the plasma membrane; and (iv) the decrease of the cholesterol dependence of P-gp ATPase activity induced by known P-gp substrates is correlated with the inhibition of the ATP-dependent cholesterol redistribution within the membrane. These data are highly evocative of a coupling between the basal ATPase activity of P-gp and its intramembrane cholesterol-redistribution function, and they are fully consistent with the possibility that P-gp may actively translocate cholesterol in the membrane. Finally, this P-gp-mediated cholesterol redistribution in the cell membrane makes it likely that P-gp contributes in stabilizing the cholesterol-rich microdomains, rafts and caveolae, and that it is involved in the regulation of cholesterol trafficking in cells.