Few reports on a biventricular working heart model with ex vivo perfusion exist owing to the complexity of establishing a circuit. Hence, we investigated it for donation after circulatory death. The heart in six juvenile pigs (~20 kg) was arrested by asphyxiation. After 30 minutes of global ischemia, the heart was harvested, reperfused with normoxemic blood cardioplegia for 20 minutes, and subsequently perfused with hyperxemic blood. After 70 minutes of controlled reperfusion, the system was switched to the biventricular working mode. Cardiac function was assessed before anoxia and during the biventricular mode. Left and right ventricular functions worsened during the biventricular mode, as compared to those before anoxia (dP/dt max , 673 ± 120 vs. 283 ± 95 and 251 ± 35 vs. 141 ± 21 mm Hg/s, respectively; P < .001). Systemic (resistance/100 g net heart weight) and pulmonary vascular resistance indexes during the biventricular mode were similar to those before anoxia (829 ± 262 vs. 759 ± 359, P = .707, and 167 ± 57 vs. 158 ± 83 dynes•sec•cm-5l-100-g net heart weight, P = .859, respectively). The biventricular working heart model with ex vivo perfusion was feasible, exhibited stable hemodynamics, and has the potential to be a powerful tool for direct cardiac function assessment.