Background: Recent studies suggest that viral interleukin 10 suppresses alloimmune response in transplantation and that cationic lipids are one of the most promising nonviral vehicles for gene therapy. The aim of this study was to examine the effect of ex vivo lipid-mediated viral ILIO gene transfer into rat lung allografts on subsequent rejection. Methods: Male F344 rats (RT11v1) underwent left lung transplantation with allografts from Brown Norway rats (RT1n). Allografts were transvascularly transfected 15 minutes after harvest with 5 mL of 1:20-diluted (group 1, n = 7) or 1:40-diluted (group 2, n = 6) GL67-pCMVievIL-10 complex. Group 3 (n = 7), serving as the control group, received I:40-diluted GL67-pCF1-chloramphenicol-acetyltransferase complex. All allografts were preserved for 3 hours at 10°C before transplantation. In all groups recipients were killed on postoperative day 5. Transgene expression of viral interleukin 10 was assessed by means of both reverse transcriptase-polymerase chain reaction and immunohistochemistry. Histologic rejection score, allograft gas exchange, exhaled nitric oxide level, and allograft cytokine mRNA expression were also assessed. Results: Dose-dependent transgene expression of viral interleukin 10 was detected by means of both reverse transcriptase-polymerase chain reaction and immunohistochemistry. Allograft gas exchange (PaO2) in groups 1 (114.06 ± 61.1 mm Hg) and 2 (108.58 ± 35.7 mm Hg) was significantly better than that in group 3 (66.4 ± 8.22 mm Hg; P = .020 and P =.023, respectively). The vascular rejection score in group 1 was significantly lower than that in group 3 (P = .032, Kruskal-Wallis test). Exhaled nitric oxide levels in group 2 (5.150 ± 6.38 ppb) were significantly lower than those in group 3 (13.517 ± 10.4 ppb; P = .039). Allograft interleukin 2 mRNA expression levels in group I (1.123 ± 0.23 relative units) were significantly lower than those in group 3 (1.753 ± 0.71 relative units; P = .038 vs group 3). Conclusions: Lipid-mediated ex vivo viral ILIO gene transfer into rat lung allografts improved graft gas exchange, reduced histologic rejection scores, downregulated graft interleukin 2 mRNA expression, and reduced exhaled nitric oxide levels by postoperative day 5. These results suggest a therapeutic potential of graft viral ILIO gene transfer as an effective immunosuppressive strategy against lung allograft rejection.