Branched and linear polyethylenimines (PEIs) are proving to be efficient, non-toxic and versatile agents for in vivo gene delivery by a number of routes. A major factor in the successful use of PEIs seems to be the small size of PEI/DNA complexes which can be achieved under controlled conditions of formulation, mainly by using PEIs of low molecular weights. This review considers the in vivo use of PEI, from formulation to delivery and analysis of gene expression. PEI delivery is already used for the analysis of numerous physiological processes. It is hoped that scrutiny of the mechanisms involved with PEI-based gene delivery at different levels of the transfection process and in different in vivo contexts will aid the transition towards its use in therapeutic situations.