BACKGROUND: Reconstruction of the right ventricular outflow tract is a frequently encountered component of many congenital cardiac repairs. We sought to tissue engineer pulmonary artery augmentation patches from retrovirally labeled endothelial progenitor and mesenchymal stem cells and determine the persistence of the seeded cells in vivo. METHODS: Autologous ovine endothelial progenitor and mesenchymal stem cells were labeled with a retroviral vector encoding green and red fluorescent proteins, coseeded onto biopolymers, and cultured for 5 days. The tissue-engineered patches were implanted into the main pulmonary artery with 1, 2, 4, and 6 week in vivo maturation (n = 8). In vivo evaluation included ultrasonography and angiography, with preimplant and explanted specimens evaluated using histologic examination and immunofluorescence. RESULTS: Echocardiography at each time demonstrated laminar pulmonary artery flow without a pressure gradient across the replaced segment. Pulmonary angiography did not exhibit stenosis or aneurysmal change. Gross appearance of all explanted patches showed progressive tissue formation with increased length of time in vivo. Retrovirally labeled cellular persistence was 96%, 82%, 85%, and 66% at 1, 2, 4, and 6 weeks after implantation, respectively. Early in the in vivo remodeling period, the number of green fluorescent protein-positive endothelial progenitor cells was 1.6 fold greater than the red fluorescent protein-positive mesenchymal stem cells. As in vivo remodeling continued, red fluorescent protein-expressing mesenchymal stem cells were expressed 1.2 to 1.7 times that of the green fluorescent protein-positive endothelial progenitor cells. CONCLUSIONS: The data demonstrate the successful creation of an anatomically functional, autologous tissue-engineered pulmonary artery using coseeded progenitor cell sources. Labeled implanted stem cells persisted in the engineered construct, suggesting that in vitro seeding is necessary to engineer tissue. This study demonstrates an effective method to track multiple cell types after implantation.