The production of viable biological heart valves is of central interest in tissue engineering (TE). The aim of this study was to generate decellularized heart valves with an intact ultra-structure and to repopulate these with endothelial cells (EC) under simulated physiological conditions. Decellularization of ovine pulmonary valve conduits was performed under agitation in detergents followed by six wash cycles. Viability of EC cultures exposed to washing solution served to prove efficiency of washing. Resulting scaffolds were free of cells with preserved extracellular matrix. Biomechanical standard tension tests demonstrated comparable parameters to native tissue. Luminal surfaces of decellularized valvular grafts were seeded with ovine jugular vein EC in dynamic bioreactors. After rolling culture for 48 h, pulsatile medium circulation with a flow of 0.1 L/min was started. The flow was incremented 0.3 L/min/day up to 2.0 L/min (cycle rate: 60 beats/min), while pH, pO2, pCO2, lactate and glucose were maintained at constant physiological levels. After 7 days, a monolayer of cells covered the inner valve surface, which expressed vWF, indicating an endothelial origin. A complete endothelialization of detergent decellularized scaffold can be achieved under simulated physiological circulation conditions using a dynamic bioreactor system, which allows continuous control of the culture environment.