Objective: We compared the efficacy of in vivo and ex vivo liposome transfection in rat lung transplantation. Methods: (1) Chloramphenicol acetyltransferase group: Fischer rats underwent isogeneic transplantation (n = 4 per group). Recipients were put to death on postoperative day 2 for chloramphenicol acetyltransferase activity. Ex vivo setting: Grafts received cDNA complexed or not with liposomes and were transplanted after 1.5 or 10 hours at 10°C. In vivo setting: Donors were intravenously injected with cDNA complexed or not with liposomes. Lungs were harvested after 1.5 or 10 hours, preserved at 10°C, and transplanted. (2) Transforming growth factor-β1 group: Brown-Norway rats served as donors and Fischer rats as recipients. All grafts were preserved for 3 hours at 10°C. On postoperative day 5, arterial oxygenation and histologic rejection scores were assessed. Ex vivo setting: Grafts received transforming growth factor-β1 sense (n = 8) or antisense (n = 7) complexed with liposomes or cDNA alone (n = 5). In vivo setting: Donors were intravenously injected with liposome:transforming growth factor-β1 sense cDNA (n = 7). Exposure time was 3 hours. Results: (1) Chloramphenicol acetyltransferase-transfection was superior in the ex vivo group but was not statistically different for longer exposure times. (2) Transforming growth factor-β1-arterial oxygenation was superior in the ex vivo liposome:sense group. cDNA alone was inefficient. Rejection scores were not statistically different between ex vivo and in vivo liposome:sense groups but were better when the ex vivo liposome:sense group was compared with the cDNA alone or the antisense groups. Conclusions: (1) With current liposome technology, the ex vivo route is superior to the in vivo approach; (2) cDNA alone does not provide transgene expression at levels to produce a functional effect.