Background: Radioiodine is efficiently concentrated by tissues expressing the human sodium iodide symporter (hNIS). Objective: To analyze the effects of iodine 131 on acute cardiac allograft rejection after ex vivo hNIS gene transfer in a rat model of cardiac allotransplantation. Materials and Methods: Hearts from Brown Norway rats were perfused ex vivo either with UW (University of Wisconsin) solution (n = 9) or UW solution containing 1 × 109 pfu/mL of adenovirus 5 plus NIS (Ad-NIS) (n = 18). Donor hearts were transplanted heterotopically into the abdomen of Lewis rats, and recipients were treated on postoperative day 3 with either 15,000 μCi of 131I or saline solution. The hearts were explanted when no longer beating, and were evaluated histologically for evidence of rejection and other changes. Results: Grafts perfused with the Ad-NIS vector survived significantly longer in recipients injected with 131I (mean [SD], 11.3 [1.9] days) compared with control animals not treated with 131I (5.7 [0.65] days) (P < .001). Treatment with 131I did not prolong graft survival in recipients of hearts that were not perfused with Ad-NIS (5.5 [1.0] vs 5.3 [0.8] days). In Ad-NIS 131I-treated transplants, the level of myocardial damage on day 6 after surgery, when control hearts were rejected, was significantly lower (60.8 [28.0] vs 99.7 [0.8]; P < .05). Conclusion: Our findings indicate that 131I, after NIS gene transfer, can effectively prolong cardiac allograft survival. To our knowledge, this is the first report of the use of NIS-targeted 131I therapy in cardiac transplantation. Further studies are required to determine the mechanism of this effect and its potential for clinical application. © 2010 Elsevier Inc. All rights reserved.