Pediatric pulmonary valves are commonly repaired by monocusp augmentation, but current repair materials cannot grow, necessitating reintervention. Heart valve tissue engineering (HVTE) may address these limitations by producing functional living materials. Here, protocols were developed for in vitro culture of monocusp tissue comprised of pig umbilical cord perivascular cells coating aligned poly-carbonate polyurethane (PCNU) membranes. The mechanical properties of PCNU membranes were influenced by sterilization method and membrane thickness, but did not change when seeded with cells and cultured for up to 28 days. In vitro hemodynamic testing of PCNU membranes as a monocusp in a pediatric sized conduit demonstrated physiological opening and closing behaviours in response to an ISO standard condition of 20 mmHg, 100 bpm, and 1.5 L/min over two hours. These studies provide new insights for the use of PCNU for HVTE and are the first demonstration of a functional in vitro tissue engineered monocusp.