Gene transfer to lung has been hindered by inflammatory and immunological responses activated to the gene-transfer agent or transgene products. In prior work, adenovirus vector delivered to the lung with the cationic glucocorticoid, dexamethasone-spermine (DS) had improved targeting to conducting airway epithelium and reduced cellular infiltration. In this study, the effect of formulation on homologous adenovirus vector re-administration was studied in C57Bl/6 mice. Formulation of an adenovirus vector expressing LacZ with DS/dioleoylphosphatidylethanolamine (DOPE) delivered at day 0 allowed re-administration of adenovirus vector expressing alkaline phosphatase at day 21. Formulation with 3beta [N-(N', N'-dimethylaminoethane) carbamoy] cholesterol (DC-Chol) DC-cholesterol (DC-Chol))/DOPE or dexamethasone in the first dosing at day 0 resulted in moderate alkaline phosphatase expression at day 24. Neutralizing antibodies against adenovirus vector in serum at day 28 were greatly reduced by all three formulations in mice receiving a single dose of adenovirus at day 0. Also, homologous adenovirus vector re-administration at day 14 produced less neutralizing antibody at day 28 when adenovirus was formulated with DS/DOPE at day 0. The use of DS/DOPE at day 0 dramatically reduced CD4 and CD8 T-cell infiltration in mice receiving adenovirus at day 0 followed by vector re-administration at day 14. Transgene-specific T-cell activation was markedly reduced by the DC-Chol/DOPE formulation. Overall, DS/DOPE) facilitated homologous vector re-administration through a combination of liposomal and glucocorticoid mechanisms.