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Based on the multilayer dielectric model for a spherical cell, a frequency response model of transmembrane potentials on cellular inner and outer membranes is established with a simulating method. The simulating results indicate that transmembrane potential on the inner membrane shows first-order bandpass filter characteristic, while transmembrane potential on the outer membrane shows first-order low-pass filter characteristic approximately. It could be found that the transmembrane potential on the inner membrane is greater than that on the outer membrane, and can keep a higher value in the range from a center frequency to an upper cutoff frequency, which is desirable to induce intracellular electromanipulation. Both a discussion about an equivalent RC model of the cell and the experimental result are in agreement with the aforementioned conclusion. Therefore, the frequency response model could help to choose reasonable window parameters for the application of a nanosecond pulsed electric field to tumor treatment.