This study deals with the development of gene therapy in the treatment of lung diseases. It reports on the use of ultrasonic nebulization to administer plasmid-lipid complexes to the lungs of mice to transfect their epithelial cells. A plasmid complexed to cationic lipids was aerosolized using an ultrasonic nebulizer. We then characterized the lipoplex size and visualized the lipoplex by electron microscopy. Finally, we assessed the in vivo transgene expression in the lungs further to the aerosolization of different lipid-plasmid formulations. The nebulizer-generated particles were small and looked like a string composed of little and more or less cubic units. Transgene expression was detected in the lungs of mice further to a 20-min exposure to aerosol particles produced with the ultrasonic nebulizer. The results obtained with our optimized plasmid-lipid-NaCl formulation suggest that this route can be used to administer an appropriate gene to the airways for the treatment of respiratory disorders.