The shifts in membrane potential, caused by the injection of D-glucose into a permeation cell, were obtained theoretically from membrane potential theory and compared to experimental data measured for immobilized (entrapped) glucose oxidase membrane and its lamellar enzyme membranes. The shifts in membrane potential were assumed to be generated by the change in charge density in the enzyme membranes in calculations. The dependence of shifts in the total membrane, Donnan, inter-Donnan and diffusion potentials on the concentration of NaCl solution in the cell was examined for some model membranes. The effective fixed-charge concentration in the enzyme membrane after the injection of D-glucose can be determined from the most negative shifts in membrane potential, since the most negative shifts depend solely on the effective fixed-charge concentration in the enzyme membrane. The diffusion path of D-glucose can be determined from the concentration of NaCl in the cell where the most negative shift was observed and it is estimated that D-glucose diffuses through 50% of an enzyme membrane of 51 µm thickness. It is suggested that the concentration of NaCl solution in the cells is important in observing the most negative shifts especially for the lamellar enzyme membrane. The theoretical curves satisfactorily explain the shifts in membrane potential obtained experimentally for not only the homogeneous enzyme membrane but also the lamellar enzyme membrane.