BACKGROUND Aerosol drug delivery currently represents the most acceptable and convenient delivery system for repeated drug application to the lungs. Although polyethyleneimine (PEI) has recently been demonstrated to mediate gene transfer successfully to mouse lungs via aerosol delivery, the effect of the jet nebulization process on the properties of PEI/DNA polyplexes has not yet been examined. METHODS PEI/DNA polyplexes were generated in several commonly used solvents, such as distilled water, HEPES buffered saline (HBS), and 5% glucose. The complex parameters, such as particle size, zeta potential, DNA integrity, and transfection efficiency, were examined before and after jet nebulization. RESULTS The complex parameters and the transfection efficiency were influenced by the solvent that was used for complex formulation and by the nebulization process itself. When polyplexes were formulated in HBS, the particle size, zeta potential, and DNA concentration were affected by the nebulization process and the transfection efficiency decreased dramatically. Polyplexes formulated in 5% glucose were less susceptible to the nebulization process, as indicated by only minor changes of the zeta potential and particle size when compared with HBS. The resulting transfecion efficiency was very low both before and after nebulization. Polyplexes formulated in distilled water had the most resistant behavior with the nebulization process. Zeta potential, particle size, and DNA integrity were influenced least of all by nebulization. CONCLUSION As a result, the transfection efficiency of PEI/DNA complexes remained constant throughout the nebulization process only when formulated in distilled water. These data suggest that distilled water represents the most appropriate solvent for polyplex formulation when delivered by jet nebulization.