Multidrug resistance P-glycoprotein (Pgp) has been reported to localize in low-density, cholesterol-enriched membranes. However, effects of low-density membrane domains on function of Pgp remain unexplored in whole cell systems. In cells that express modest levels of the protein endogenously or through drug selection, Pgp predominantly localized to low-density membranes following separation on a sucrose gradient. When highly overexpressed in NIH 3T3 cells, a prominent amount of Pgp also was detected in high-density membranes. Removing cholesterol from cells with beta-methylcyclodextrin (CD), a sterol acceptor molecule, shifted fractions that contained Pgp from low toward high density, and this effect was reversed to a similar extent by restoring sterols with either cholesterol or enantiomeric cholesterol. However, function of human MDR1 Pgp as probed with Tc-Sestamibi, a transport substrate for Pgp, was not dependent on localization of Pgp in cholesterol-enriched membranes. Specific inhibition of MDR1 Pgp with GF120918 or LY335979 also was independent of cholesterol. Cell-type-specific effects of cholesterol content on function of human Pgp were detected by use of daunomycin, another substrate for Pgp, although efficacy of inhibitors remained independent of cholesterol. Conversely, both function and inhibition of hamster Pgp as measured with Tc-Sestamibi and daunomycin were in part dependent on normal cell content of cholesterol. These data show that Pgp preferentially localizes to low-density, cholesterol-enriched membrane domains, but acute depletion of cholesterol impacts Pgp-mediated drug transport in a substrate- and cell-type-specific manner.