Background: Adenovirus serotype 5 has remained the pre-eminent vector in pre-clinical gene therapy applications in cardiac transplantation. Concerns over the potential effects of adenoviral vectors on the later development of cardiac allograft vasculopathy (CAV) are addressed in this study. Methods: Hearts (n = 22) harvested from Brown Norway rats were perfused ex vivo with either University of Wisconsin (UW) solution with no virus, Ad-CMV-LacZ or Ad-CMV-Null. Donor hearts were transplanted heterotopically into the abdomen of Lewis rats. All recipients received cyclosporine for the duration of the experiment. Transplanted hearts were recovered for analysis at 120 days. Sections of the heart were stained with elastic-van Gieson stain for morphometric analysis of the vessels to ascertain the degree of vascular luminal occlusion. Hematoxylin-eosin staining facilitated diagnosis of chronic rejection. Results: Seventy-seven percent of transplanted hearts showed signs of chronic rejection with no difference in the proportion of animals between groups (p = 0.797). No difference was noted in the degree of vascular luminal occlusion between the Ad-Null (0.57 ± 0.22), Ad-LacZ (0.62 ± 0.19) and UW (0.47 ± 0.29) groups (p = 0.653). Conclusions: Vascularized cardiac allografts transplanted from Brown Norway to Lewis rats demonstrated cardiac allograft vasculopathy CAV at 120 days. Adenoviral perfusion of the donor heart ex vivo did not affect the development of CAV.