Our previous studies revealed that the dielectrophoresis method is effective for separating cells having different dielectric properties. The purpose of this study was to evaluate the separation characteristics of two kinds of cells by direct current (DC) voltage offset/alternating current (AC) voltage using an insulating porous membrane dielectrophoretic separator. The separation device gives dielectrophoretic (DEP) force and electrophoretic (EP) force to dispersed particles by applying the DC-offset AC voltage. This device separates cells of different DEP properties by adopting a structure in which only the parallel plate electrodes and the insulating porous membrane are disposed in the flow path through which the cell-suspension flows. The difference in the retention ratios of electrically homogeneous 4.5 m or 20.0 m diameter standard particles was a maximum of 82 points. Furthermore, the influences of the AC voltage or offset voltage on the retention ratios of mouse hybridoma 3-2H3 cells and horse red blood cells (HRBC) were investigated. The difference in the retention ratio of the two kinds of cells was a maximum of 56 points. The separation efficiency of this device is expected to be improved by changing the device shape, number of pores, and pore placement. (c) 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1292-1300, 2016