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This study features the longest experimental follow-up for decellularized heart valves implanted in an animal model. Porcine aortic heart valves were decellularized according to a disclosed standardized method in which TRITON X-100 and sodium cholate (TRICOL) are used in succession, followed by a further treatment with the endonuclease Benzonase to completely remove the nucleic acid remnants. Experimental animals ( n = 17), represented by Vietnamese pigs (VPs), received a decellularized aortic allograft as a substitute for the replacement of their right ventricular outflow tract. The surgical implantation of the TRICOL-treated aortic valve conduit was successful in 11 VPs, while perioperative or postoperative complications occurred in the remaining six animals. In the sham-operated group ( n = 4), the native pulmonary root was excised and immediately reimplanted orthotopically in the same animal. Echocardiography demonstrated a satisfactory hemodynamic performance of the TRICOL-treated valves during follow-up as well as the absence of relevant leaflet alterations concerning thickness and motility or valve insufficiency. At explantation, macroscopic inspection of tissue-engineered heart valve conduits did not evidence calcifications and showed a decreased wall thickness, comparable to that of the reimplanted native pulmonary roots. Noteworthy, extended functional performance, recovery of DNA content, and active extracellular matrix precursor incorporation are apparently compatible with the properties of a living self-supporting substitute. [ABSTRACT FROM AUTHOR]