Nitric oxide synthase (NOS) inhibitors have been shown to reduce NO but yield conflicting results on cardiac allograft survival. In this study, we provide an alternative approach specifically to examine the efficacy of a NO scavenger on nitrosyl complex formation and graft survival in a model of heterotopic cardiac transplantation. Efficacy was examined under both acute and chronic conditions (i.e., without or with immunosuppression, respectively). Electron paramagnetic resonance (EPR) spectroscopy of frozen myocardial tissue from untreated allografts showed progressive increases in nitrosylheme and nitrosomyoglobin before graft failure. These signals were not seen in either isografts or native hearts of allograft recipients. Both plasma nitrate plus nitrite and myocardial nitrosyl complex formation in cardiac allografts were significantly decreased in recipient animals treated with the NO scavenger, NOX-100, or by low-dose cyclosporine (CsA). Both interventions were nearly equivalent in significantly prolonging graft survival. The short-term combination treatment of both NOX-100 plus CsA completely eliminated myocardial nitrosyl complex formation and synergistically prolonged graft survival. Long-term combination drug treatment (days 0-100) followed by cessation of therapy resulted in permanent graft acceptance with no evidence for nitrosyl complex formation. These studies support a role of NO in cardiac allograft rejection. Furthermore, these studies indicate a potential therapeutic value of NO scavengers in preventing organ rejection.