Viruses have long been considered to be the most promising tools for human gene therapy. However, the initial enthusiasm for the use of viruses has been tarnished in the light of potentially fatal side effects. Transposons have a long history of use with bacteria in the laboratory and are now routinely applied to eukaryotic model organisms. Transposons show promise for applications in human genetic modification and should prove a useful addition to the gene therapy tool kit. Here we review the use of viruses and the limitations of current approaches to gene therapy, followed by a more detailed analysis of transposon length and the physical properties of internal sequences, which both affect transposition efficiency. As transposon length increases, transposition decreases: this phenomenon is known as length-dependence, and has implications for vector cargo capacity. Disruption of internal sequences, either via deletion of native DNA or insertion of exogenous DNA, may reduce or enhance genetic mobility. These effects may be related to host factor binding, essential spacer requirements or other influences yet to be elucidated. Length-dependence is a complex phenomenon driven not simply by the distance between the transposon ends, but by host proteins, the transposase and the properties of the DNA sequences encoded within the transposon.