Electropermeabilization is a promising nonviral method for gene therapy. However, despite the fact that it is widely used to transfer genes into living cells, the steps that limit DNA transfer remain to be determined. Here, we report the effect of cell synchronization on membrane permeabilization and gene delivery by electric fields. Chinese hamster ovary (CHO) cells were synchronized by aphidicolin or butyrate treatment. Electro-mediated transfection of these cells was evaluated under electric field conditions leading to the same level of membrane permeabilization. Aphidicolin cell synchronization in G2/M phase leads to a slight increase in plasma membrane permeabilization but to a three-fold increase in percentage of transfected cells and to an eight-fold increase in gene expression. This increase in cell transfection is specifically due to the G2/M synchronization process. Indeed, cell synchronization in G1 phase by sodium butyrate has no effect on cell permeabilization and transfection. Our results suggest that the enhanced transfection level in G2/M phase is not simply due to enhanced permeabilization, but reinforce the statement that the melting of the nuclear membrane facilitates direct access of plasmid DNA to the nucleus.