Selective separation of cells using dielectrophoresis (DEP) has recently been studied and methods have been proposed. However, these methods are not applicable to large-scale separation because they cannot he performed efficiently. In DEP separation, the DEP force is effective only when it is applied close to the electrodes. Utilizing a DEP filter is a solution for large-scale separation. In this article, the separation efficiency for viable and nonviable cells in a DEP filter was examined. The effects of an applied AC electric field frequency and the gradient of the squared electric field intensity on a DEP velocity for the viable and nonviable animal cells (3-2H3 cell) were discussed. The frequency response of the DEP velocity differed between the viable and the nonviable cells. We deducted an empirical equation that can be used as guiding principle for the DEP separation. The results indicate that the viable and the nonviable cells were separated using the DEP filter, and the best operating conditions such as the applied voltage and the flow rate were discussed. (C) 2010 American Institute of Chemical Engineers Biotechnol. Prog., 26: 1061-1067, 2010