The shifts in membrane potential, caused by the injection of glucose into a permeation cell, were measured using immobilized (entrapped) glucose oxidase membranes. No pH change in the permeation cell was observed upon injection of glucose, but the shift in membrane potential was definitely detected. The shift in membrane potential was observed under nitrogen bubbling (in the absence of oxygen) using initially used enzyme membranes. It was, therefore, suggested that the shifts in membrane potential were not caused by an enzyme-substrate reaction, but by binding of the substrate to the enzyme, which induces a conformational change in the enzyme and leads to a change in charge density in the enzyme membrane. This mechanism is also supported by the fact that the shifts in membrane potential were observed upon injection of not only D-glucose but also L-glucose as reported in our previous study [ J. Chem. Soc. Faraday Trans., 87, 695 (1991) 1.