Gene transfer to humans requires carriers for the plasmid DNA which can efficiently and safely carry the gene into the nucleus of the desired cells. A series of chemically different cationic polymers are currently being investigated for these purposes. Although many cationic polymers indeed condense DNA spontaneously, which is a requirement for gene transfer in most types of cells, the physicochemical and biopharmaceutical behavior of the current generation of polyplexes severely limits an efficient gene transfer in vitro and especially in vivo. This paper summarizes recent physicochemical and biological information on polyplexes and aims to provide new insights with respect to this type of gene delivery system. Firstly, the chemical structure of frequently studied cationic polymers is represented. Secondly, the parameters influencing condensation of DNA by cationic polymers are described. Thirdly, the surface properties, solubility, aggregration behavior, degradation and dissociation of polyplexes are considered. The review ends by describing the in vitro and in vivo gene transfection behavior of polyplexes.