Spray-drying is an effective process for preparing micron-dimensioned particles for pulmonary delivery. Previously, we have demonstrated enhanced dispersibility and fine particle fraction of spray-dried nonviral gene delivery formulations using amino acids or absorption enhancers as dispersibility-enhancing excipients. In this study, we investigate the use of the cationic polymer chitosan as a readily available and biocompatible dispersibility enhancer. METHODS: Lactose-lipid:polycation:pDNA (LPD) powders were prepared by spray-drying and post-mixed with chitosan or spray-dried chitosan. In addition, the water-soluble chitosan derivative, trimethyl chitosan, was added to the lactose-LPD formulation before spray-drying. RESULTS: Spray-dried chitosan particles, displaying an irregular surface morphology and diameter of less than 2 μm, readily adsorbed to lactose-LPD particles following mixing. In contrast with the smooth spherical surface of lactose-LPD particles, spray-dried trimethyl chitosan-lactose-LPD particles demonstrated increased surface roughness and a unimodal particle size distribution (mean diameter 3.4 μm), compared with the multimodal distribution for unmodified lactose-LPD powders (mean diameter 23.7 μm). The emitted dose and in vitro deposition of chitosan-modified powders was significantly greater than that of unmodified powders. Moreover, the inclusion of chitosan mediated an enhanced level of reporter gene expression. CONCLUSIONS: In summary, chitosan enhances the dispersibility and in vitro pulmonary deposition performance of spray-dried powders.