Perfusion decellularization of cadaveric hearts generates a cell-free extracellular matrix (ECM) that has similar architectural, mechanical, and chemical properties to the native tissue. These scaffolds retain their vascular conduits giving them the potential for anastomosis and transplantation. However, because the matrix is denuded, vascular clotting occurs after transplant even under an anti-coagulation (Coumadin) regimen. To create a less thrombogenic and thus more transplantable decellularized construct, we re-lined vessel conduits throughout the heart with endothelial cells and quantified their functional ability to alter clotting. Cadaveric rat hearts were decellularized by retrograde aortic detergent perfusion. Labeled rat aortic endothelial cells (RAECs) were re-introduced following two different protocols: 40 million RAECs were delivered by arterial (Ar) infusion only, or 20 million RAECs delivered by Ar infusion followed with an additional 20 million RAECs delivered by venous (Ve) infusion. Re-endotheliazed heart constructs were then maintained in a bioreactor under retrograde aortic perfusion for 1 week. Thrombogenicity of the constructs was measured by an in vitro thrombomodulin assay of the media flowing through the heart vasculature followed by histologic analysis. Heterotopic transplantation of scaffolds was performed in Rowett Nude rats, and hearts were examined 1 week post transplant. Either delivery route resulted in a whole heart (base to apex) distribution of RAECs with the retention of the endothelial phenotype (e.g. flattened morphology, proliferative (PCNA + ), eNOS + and vWF + ). In vitro thrombogenicity was reduced regardless of the delivery protocol, though there was a trend towards improved performance when cells were delivered via both Ar+Ve routes (6 and 8 fold decrease vs. controls for Ar only or Ar+Ve delivery respectively, N=8 for each condition, p<0.05). After heterotopic transplantation of the re-endothelialized ECM, clotting and cellular invasion were reduced. With this advance we move closer to having a suitable heart construct for transplant while also developing a novel in vitro system for use in studies such as examining stem/progenitor cell differentiation in decellularized heart.