Objective: Lung transplantation is limited by the scarcity of donor organs. Lung retrieval from non-heart-beating donors (NHBD) might extend the donor pool and has been reported recently. However, no studies in NHBD exist using the novel approach of retrograde preservation with Perfadex solution. Methods: Heparinized asystolic pigs (n = 5, 30–35 kg) were ventilated for 90 min. The lungs were retrogradely preserved with Perfadex solution and stored inflated at 4°C for 3 h. Left lung transplantation in the recipient was followed by exclusion of the right lung. Results were compared to sham-operated animals. Oxygenation, hemodynamics and dynamic compliance were monitored for 4 h. Infiltration of polymorphonuclear cells (PMNs) and stereological quantification of alveolar edema was performed. Statistical analysis comprised Kruskal-Wallis and Mann-Whitney tests and ANOVA analysis with repeated measures. Results: No mortality was observed. During preservation, continuous elimination of blood clots via the pulmonary artery venting site was observed. Oxygenation and compliance were similar between groups, but sham controls showed significantly lower pulmonary vascular resistance. Stereological quantification revealed higher volume fractions of intra-alveolar edema in NHBD grafts, while PMN infiltration was comparable to sham controls. Conclusions: Use of NHBD lungs results in excellent outcome after 90 min of warm ischemia followed by retrograde preservation with Perfadex solution. This novel approach can optimize lung preservation by eliminating clots from the pulmonary circulation and might clinically be considered in brain-dead organ donors who become hemodynamically unstable prior to organ harvest. Further trials with longer warm and cold ischemic periods are necessary to further elucidate this promising approach to donor pool expansion.