Objective: Multiple gene transfer might permit modulation of concurrent biochemical pathways involved in lung graft ischemia-reperfusion injury. In this study we analyzed whether recipient intramuscular naked plasmid cotransfection of transforming growth factor β1 and interleukin 10 would result in amelioration of lung graft ischemia-reperfusion injury. Methods: Forty-eight hours before transplantation, 6 groups (n = 6) of F344 rats received intramuscular injection of naked plasmid encoding chloramphenicol acetyltransferase, chloramphenicol acetyltransferase plus β-galactosidase, transforming growth factor β1, interleukin 10, or transforming growth factor β1 plus interleukin 10 or were not treated. Donor lungs were flushed and stored for 18 hours at 4°C before transplantation. Twenty-four hours later, grafts were assessed immediately before the animals were killed. Arterial oxygenation, wet/dry ratio, myeloperoxidase, and proinflammatory cytokines (interleukin 1, tumor necrosis factor α, interferon γ, and interleukin 2) were measured, and immunohistochemistry was performed. Results: For lung graft function, the arterial oxygenation was considerably higher in the cotransfected group receiving transforming growth factor β1 plus interleukin 10 compared with that in all other groups (P ≤ .03). The wet/dry ratio, reflecting lung edema, was reduced in the cotransfected group compared with that in control animals (nontreated, P < .02; chloramphenicol acetyltransferase, P < .03; chloramphenicol acetyltransferase plus β-galactosidase, P < .01). Myeloperoxidase, which measures neutrophil sequestration, was also reduced with cotransfection compared with that seen in control animals (P ≤ .03). All proinflammatory cytokines were decreased in the cotransfected group compared with those in all other groups (interleukin 1β, P < .04; tumor necrosis factor α, P < .002; interferon γ, P < .0001; interleukin 2, P < .03). These results indicate that cotransfection provides a synergistic benefit in graft function versus either cytokine alone, neutrophil sequestration, or inflammatory cytokine expression. Immunohistochemistry showed positive staining of transforming growth factor β1 plus interleukin 10 in type I and II pneumocytes and localized edema fluid. Conclusions: Recipient intramuscular naked plasmid cotransfection of transforming growth factor β1 and interleukin 10 provides a synergistic effect in ameliorating lung reperfusion injury after prolonged ischemia.