Until recently, the attention of most researchers has focused on the first and last steps of gene transfer, namely delivery to the cell and transcription, in order to optimise transfection and gene therapy. However, over the past few years, researchers have realised that the intracellular trafficking of plasmids is more than just a "black box" and is actually one of the major barriers to effective gene delivery. After entering the cytoplasm, following direct delivery or endocytosis, plasmids or other vectors must travel relatively long distances through the mesh of cytoskeletal networks before reaching the nuclear envelope. Once at the nuclear envelope, the DNA must either wait until cell division, or be specifically transported through the nuclear pore complex, in order to reach the nucleoplasm where it can be transcribed. This review focuses on recent developments in the understanding of these intracellular trafficking events as they relate to gene delivery. Hopefully, by continuing to unravel the mechanisms by which plasmids and other gene delivery vectors move throughout the cell, and by understanding the cell biology of gene transfer, superior methods of transfection and gene therapy can be developed.