The combined use of the membrane surface potential fluorescent sensor fluorescein phosphatidylethanolamine (FPE) and the membrane dipole potential fluorescent sensor di-8-ANEPPS to characterize the interaction of molecules with model and cellular membranes and to asses the influence of the dipole potential on the interaction is reported. The study of the human immunodeficiency virus protease inhibitor saquinavir with Caco-2 cells and phospholipid membranes reveals that the compound interacts with the lipidic bilayer of model membranes with a simple hyperbolic binding profile but with Caco-2 cells in a cooperative way involving membrane receptors. Additional studies indicated that colchicine acts as a competitor ligand to saquinavir and suggests, in agreement with other reports, that the identity of the saquinavir "receptor" could be P-glycoprotein or the multiple drug resistance-associated protein. The modification of the magnitude of the membrane dipole potential using compounds such as cholesterol, phloretin, and 6-ketocholestanol influences the binding capacity of saquinavir. Furthermore, removal of cholesterol from the cell membrane using methyl-beta-cyclodextrin significantly decreases the binding capacity of saquinavir. Because removal of cholesterol from the cell membrane has been reported to disrupt membrane domains known as "rafts," our observations imply that the membrane dipole potential plays an important role as a modulator of molecule-membrane interactions in these membrane structures. Such a role is suggested to contribute to the altered behavior of receptor-mediated signaling systems in membrane rafts.