Objective: Because almost all pulmonary diseases are not caused by one gene, multiple gene transfection is required for current gene therapy. Adenovirus is an important gene therapy vector, but a short duration and the inability of repeated administration remain limitations. The aims of this study were to evaluate whether adenoviral vector encoding soluble tumor necrosis factor a receptor immunoglobulin and β-galactosidase cotransfection prolongs gene expression and facilitates repeated vector administration to investigate the feasibility of a cotransfection strategy. Methods: F344 rats received intratracheal administration of 1 × 109 plaque-forming units of adenoviral vector encoding β-galactosidase or both adenoviral vector encoding β-galactosidase and adenoviral vector encoding soluble tumor necrosis factor α receptor immunoglobulin. In the expression study β-galactosidase gene expression in the lung was examined by means of enzyme-linked immunosorbent assay on days 2, 7, 14, 28, and 56 (n = 4/day). In the repeated transfection study, soluble tumor necrosis factor α receptor immunoglobulin and β-galactosidase were readministered once (7 days after the first adenovirus administration) or twice (on days 7 and 14; n = 4/day). A 2-way factorial analysis of variance was used for statistical analysis. Results: Soluble tumor necrosis factor α receptor immunoglobulin and β-galactosidase cotransfection prolonged the duration of β-galactosidase expression. However, antiadenovirus antibody production was significantly increased in the cotransfection group. In addition, there was no increase in β-galactosidase expression after readministration of soluble tumor necrosis factor α receptor immunoglobulin and β-galactosidase. Conclusion: Adenoviral vector encoding soluble tumor necrosis factor a receptor immunoglobulin and β-galactosidase cotransfection prolongs β-galactosidase expression but does not increase β-galactosidase expression after repeated administration. These results suggest that tumor necrosis factor a is one of the most important factors in regulating the duration of gene expression. The cotransfection approach is feasible, but the increase of antiadenovirus antibodies might make repeated cotransfection unfeasible.