Gene electrotransfer is a promising non-viral method of gene delivery. In our in vitro study we addressed open questions about this multistep process: how electropermeabilization is related to electrotransfer efficiency; the role of DNA electrophoresis for contact and transfer across the membrane; visualization and theoretical analysis of DNA-membrane interaction and its relation to final transfection efficiency; and the differences between plated and suspended cells. Combinations of high-voltage and low-voltage pulses were used. We obtained that electrophoresis is required for the insertion of DNA into the permeabilized membrane. The inserted DNA is slowly transferred into the cytosol, and nuclear entry is a limiting factor for optimal transfection. The quantification and theoretical analysis of the crucial parameters reveals that DNA-membrane interaction (NDNA) increases with higher DNA concentration or with the addition of electrophoretic LV pulses while transfection efficiency reaches saturation. We explain the differences between the transfection of cell suspensions and plated cells due to the more homogeneous size, shape and movement of suspended cells. Our results suggest that DNA is either translocated through the stable electropores or enters by electo-stimulated endocytosis, possibly dependent on pulse parameters. Understanding of the mechanisms enables the selection of optimal electric protocols for specific applications.