The lung is an important target of gene therapeutic interventions. In contrast to intratracheal instillation, inhalation would be the most practical route of administration in clinical applications. Here we show that aerosolized nanogram quantities of pDNA complexed to PEI (350 ng) yielded transfection levels 15-fold higher than a 140-fold higher dose (50 microg) of the same vector applied directly to the lungs of mice via intratracheal intubation. An important efficacy parameter is the osmolarity of the aerosol and not biophysical properties of the nebulized vector. Vectors formulated and nebulized in hypoosmotic distilled water yielded 57- and 185-fold higher expression levels than those in isotonic 5% glucose or Hepes-buffered saline, respectively. Pretreatment of mice with nebulized indomethacin, which prevents water-induced airway alteration, resulted in lower gene expression, whereas pretreatment with EGTA or polidocanol, which modulate tight-junction activity, had no effect. These results, together with histological analysis of regional lung deposition and gene expression, suggest that a temporary water-induced hypoosmotic shock permeabilizes the epithelium sufficiently to allow vector uptake. The so far observed inefficiency of nonviral gene delivery to the airways may be the result of an inappropriate method of vector administration.