Abstract The chronic organ shortage has led to the reconsideration of marginal donor pools such as non–heart-beating donors (NHBD). The use of these livers is limited due to their minimal tolerance for cold preservation. The aim of this study was to examine the combination of two different therapeutic strategies for the preservation of livers from NHBD. The livers of male Wistar rats were harvested after the induction of cardiac arrest via phrenotomy (30, 90 minutes). Livers were perfused with 10 mL of UW solution (UW), followed by hypothermic preservation with or without insufflation of gaseous oxygen (O 2). In one group a fibrinolytic preflush (10 mL of Ringer's containing 7500 IU of streptokinase) was performed with subsequent preservation with O 2 (O 2+SK). After storage (24 h/4°C/UW) livers were reperfused in vitro. Livers retrieved from heart beating donors served as controls. The results showed that even after only 30 minutes of warm ischemia livers displayed a serious disturbance in vascular perfusion (portal venous pressure, PVP = 7.4 ± 0.2* versus control: 4.1 ± 0.5 mm Hg), associated with a more than 10-fold increase in enzyme release (ALT: 26819 ± 513* versus control 683 ± 152 mU/g/L), which was consistent with a significant depression in bile synthesis (1.21 ± 0.35* versus 19.36 ± 2.16 μL/g/45 min). However, these impairments could be prevented with O 2. Even after 90 minutes of WI, the function was significant better using aerobic preservation (ALT: 3204 ± 549 mU/g/L). With a supplementary fibrinolytic preflush, we effectively preserved livers up to 90 minutes of WI with results comparable to livers from heart beating donors with no WI (ALT: 1623 ± 432 mU/g/L). The combination of these two techniques represents a new therapeutic approach for livers with extended or unclear WI periods in non–heart-beating donors (* P < .05 versus control).