Nebulisation is currently the most acceptable and practical delivery system for repeated applications of gene therapy to the lower airways of cystic fibrosis (CF) patients. We have assessed whether this route of administration offers other benefits with regard to respiratory gene transfer. A standard jet nebuliser (Acorn System 22, Medicaid) was used to transfer the reporter gene beta-galactosidase complexed with the cationic liposome DC-Chol/DOPE to three epithelial cell lines in vitro, two non-CF and one CF, using a novel collection system. In all three cell lines, nebulisation resulted in significantly (P < 0.05) improved transfection efficiency compared with instillation. At a constant DNA: liposome ratio of 1:5 (wt:wt), transfection efficiency was inversely related to increasing concentrations of DNA-liposomes before nebulisation. This effect was not related to the amount of DNA delivered and measurements of both zeta potential and mean aerodynamic particle size before and after nebulisation did not show concentration-related differences. The increased transfection efficiency did not relate either to the physical consequences of the nebulisation processes nor the effects of nebulisation on the complexes before instillation. Significantly increased transfection efficiency was seen following nebulisation with 95% O2/5% CO2 in comparison with 21% O2/78% N2 (air); this did not relate to changes in either the pH or temperature of the solution bathing the cells. The data confirm that nebulisation is appropriate for gene delivery to the lower airways in clinical practice and points to factors that may optimise gene transfer efficiency.