The phenomenon of multidrug resistance (MDR) is reinterpreted on the basis of the recently proposed concept of microvillar signaling. According to this notion, substrate and ion fluxes across the surface of differentiated cells occur via transporters and ion channels that reside in membrane domains at the tips of microvilli (MV). The flux rates are regulated by the actin-based cytoskeletal core structure of MV, acting as a diffusion barrier between the microvillar tip compartment and the cytoplasm. The expression of this diffusion barrier system is a novel aspect of cell differentiation and represents a functional component of the natural defense system of epithelial cells against environmental hazardous ions and lipophilic compounds. Because of the specific organization of epithelial Ca²⁺ signaling and the secretion, lipophilic compounds associated with the plasma membrane are transferred from the basal to the apical cell surface by a lipid flow mechanism. Drug release from the apical pole occurs by either direct secretion from the cell surface or metabolization by the microvillar cytochrome P-450 system and efflux of the metabolites and conjugation products through the large multifunctional anion channels localized in apical MV. The natural microvillar defense system also provides a mechanistic basis of acquired MDR in tumor cells. The microvillar surface organization is lost in rapidly growing cells such as tumor or embryonic cells but is restored during exposure of tumor cells to cytotoxins by induction of a prolonged G₀/G₁ resting phase.