A major issue in congenital heart surgery is the lack of viable right ventricular outflow tract (RVOT) replacement materials. Several biomaterials have been used, with different scaffolds and cells, but they have failed to restore a tri-layered RVOT, and reoperations are often required. We investigated the function, histological changes and potential of growth and tissue regeneration of polydioxanone (PDO) electrospun bioabsorbable valved patches seeded with mesenchymal stem cells (MSCs) in the RVOT of growing lambs. Autologous blood-derived MSCs were labeled with quantum dots and seeded on PDO electrospun valved patches. Those were implanted into the RVOT of 6 growing lambs followed up until 8 months. Results were assessed by echocardiography, magnetic resonance imaging (MRI), histology, immunohistochemistry and biochemical assays. Tissue-engineered RVOT were neither stenotic nor aneurismal and displayed a growth potential, with less fibrosis, less calcifications and no thrombus compared with control polytetrafluoroethylene (PTFE)-pericardial patches. The PDO scaffold was completely degraded and replaced by a viable, three-layered, endothelialized tissue and an extracellular matrix with elastic fibers similar to that of native tissue. Detection of quantum dots at 1 month suggested that at least some of the cells were-derived from the grafted cells. A polydioxanone electrospun tissue-engineered valved transannular patch seems to be a promising device in restoring a living RVOT and could ultimately lead to applications in the treatment of congenital RVOT diseases. © 2010 Elsevier Ltd. All rights reserved.