Nanoparticles containing DNA compacted with poly-l-lysine modified on an N-terminal cysteine with polyethylene glycol can effectively transfect cells of the airway epithelium when applied by the luminal route. To evaluate the toxicity of these nanoparticles, we administered 10 and 100 μg DNA compacted into nanoparticles suspended in normal saline by the intranasal route to mice and determined the pulmonary and systemic responses to this challenge, compared to administration of saline alone, and in some experiments, compared to administration of naked DNA, Escherichia coli genomic DNA, or lipofectin-complexed naked DNA. There was no systemic response to either dose of nanoparticles in serum chemistries, hematologic parameters, serum complement, IL-6, or MIP-2 levels or in the activity, growth, and grooming of the mice. Nanoparticles containing 10 μg DNA induced responses comparable to saline in all measures, including BAL cell counts and differentials and cytokine levels and histology. However, mice dosed with 100 μg DNA in nanoparticles had modest increases in BAL neutrophils 48 and 72 h after dosing, modest increases in BAL IL-6 and KC beginning 24 and 48 h, respectively, after dosing, and, on histology of the lung, a trace to 1+ mononuclear cell infiltrates about the pulmonary veins at 48 h, which were markedly reduced by 10 days and gone by 28 days after dosing. BAL neutrophil and cytokine responses were no greater than those entrained by naked DNA for up to 24 h. However, compared to administration of only 10 μg E. coli genomic DNA, the response to compacted DNA was much less. A low dose of lipofectin-complexed DNA (5 μg DNA) induced the same response as 20-fold higher doses of DNA nanoparticles. These data indicate that DNA nanoparticles have no measurable toxic effect at a dose of 10 μg and a very modest effect, which is not limiting, at a dose of 100 μg, which gives maximal gene expression. This favorable toxicity profile encourages development of stabilized compacted DNA for airway administration.