Nonviral vectors are increasingly attracting interest as tools for the transfer of genetic material into cells. [1] Compared to viral gene-transfer systems, they offer several advantages, especially flexibility regarding the size and type of nucleic acid to be transported, biosafety, and a chemically controllable structure. Based on different chemical building blocks, a broad variety of nonviral vectors have been designed. Polyethylenimines (PEIs) are especially attractive polymers for gene delivery because their high buffering capacity promotes endosomal release, thereby favoring high in vitro and in vivo transfection efficiencies of the PEI/DNA polyplexes. [2] Current efforts in polyplex synthesis are aimed at further optimizing the transfection efficiencies. Insight into the behavior of polyplexes in live cells is a prerequisite for the development of new strategies and their validation. Fluorescence microscopy is an ideal tool for the elucidation of the respective mechanisms. Previous experiments gave the first insights into the interaction of polyplexes with negatively charged proteoglycans at the cell surface. [3,4] However, little is known about the transport processes that allow the polyplexes to overcome the barriers occurring between binding to the cell surface and the final transcription of the transgene. To unravel the dynamics of the processes involved, new singleparticle tracking techniques are employed. [5][6][7] First results from such experiments indicate that, once inside the cell, [