Electropermeabilization is a physical method to deliver molecules into cells and tissues. Clinical applications have been successfully developed for antitumoral drug delivery and clinical trials for gene electrotransfer are currently underway. However, little is known about the mechanisms involved in this transfer. The main difficulties stem from the lack of single cell models which reliably replicate the complex in vivo environment. In order to increase our understanding of the DNA electrotransfer process, we exploited multicellular tumor spheroids as an ex vivo model of tumor. We used confocal microscopy to visualize the repartition of permeabilized cells in spheroids subjected to electric pulses. Our results reveal that even if cells can be efficiently permeabilized with electric fields, including those cells present inside the spheroids, gene expression is by contrast limited to the external layers of cells. Taken together, these results, in agreement with the ones obtained in tumors, indicate that the spheroid model is more relevant to an in vivo situation than cells cultured as monolayers. They validate the spheroid model as a way to study electro-mediated gene delivery processes.