The membrane potential is mainly maintained by the K(+) concentration gradient across the cell membrane between the cytosol and the extracellular matrix. Here, we show that extracellular addition of high-molecular weight hyaluronan depolarized the membrane potential of human fibroblasts, human embryonic kidney cells (HEK), and central nervous system neurons in a concentration-dependent manner, whereas digestion of cell surface hyaluronan by hyaluronidase caused hyperpolarization. This effect could not be achieved by other glycosaminoglycans or hyaluronan oligosaccharides, chondroitin sulfate, and heparin which did not affect the membrane potential. Mixtures of high-molecular weight hyaluronan and bovine serum albumin had a larger depolarization effect than expected as the sum of both individual components. The different behavior of high-molecular weight hyaluronan versus hyaluronan oligosaccharides and other glycosaminoglycans can be explained by a Donnan effect combined with a steric exclusion of other molecules from the water solvated chains of high-molecular weight hyaluronan. Depolarization of the plasma membrane by hyaluronan represents an additional pathway of signal transduction to the classical CD44 signal transduction pathway, which links the extracellular matrix to intracellular metabolism.