To achieve effective plasmid-based gene therapy, the control of cellular access and uptake, intracellular trafficking and nuclear retention of plasmids must be achieved. Inefficient endosomal release, cytoplasmic transport and nuclear entry of plasmids are amongst some of the key limiting factors in the use of plasmids for effective gene therapy. A number of non-viral gene delivery systems have been designed to overcome these limiting factors. The most common approach to protect and control plasmid distribution is to complex plasmids with cationic lipids or polymers through electrostatic interactions. Endosomal release of plasmids can be achieved, for instance, by using pH-sensitive lipids, inactivated viral particles, endosomolytic peptides and polymers. Among the least explored gene delivery systems are those that consist mainly of synthetic, short peptides. Peptides can be incorporated into multicomponent gene delivery complexes for specific purposes, such as for DNA condensation, cell-specific targeting, endosomolysis or nuclear transport. The aims of this review are to: (i) explore the conceptual and experimental aspects of peptide-DNA interactions; (ii) critically assess the possible use of peptides for efficient gene transfer; and (iii) present an overview on the use of peptides to enhance the effectiveness of other gene delivery systems. On balance, peptide-based gene delivery systems appear to have a significant potential as commercially viable gene delivery products.