An experimental method has been established to measure the electric properties of a cell membrane by combination of patch clamp and dual-wavelength ratio imaging of a fluorescent potentiometric dye, 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octylamino)-6-naphthyl]vinyl ]pyridinium betaine (di-8-ANEPPS). Pairs of fluorescence images from the dye-stained membrane of neuroblastoma N1E-115 cells excited at two wavelengths were initially obtained to calculate ratio images corresponding to the resting transmembrane potential. Subsequently, a whole-cell patch was established and the membrane potential clamped to levels varying from -100 to +60 mV; at each voltage, a pair of dual-wavelength images were acquired to develop a calibration of the fluorescence ratio. Using this method, the resting potentials could accurately be measured showing that the differentiated cells were 17 mV more polarized than undifferentiated cells. The combination of electrical and optical methods can also follow changes in other membrane electric properties, such as dipole potential, and thus permit a detailed analysis of the membrane electrical properties underlying the voltage regulation of ion channels.