Recently, much attention has focused on the use of hypothermic perfusion in isolated organs (Hessheimer, 2012). Preservation of livers at hypothermia (20 °C) lead to significant improvements (Vairetti, 2009) but also induces oxidative stress (Alva, 2013). The initial protection against oxidative damage may require the rapid removal of oxidized proteins by the ubiquitin-proteasome system (UPS). Whereas there is some consensus in that proteasomal activity degrades oxidized proteins, it remains controversial whether oxidized proteins are tagged by ubiquitin and then degraded by the 26S proteasome, or whether they are removed independently of ubiquitin (Shang, 2011). The aim of the present work was to study the production of oxidative stress and the role of the UPS after hypothermic perfusion in isolated liver. The procedure was approved by the Committee of Animal Care following EC guidelines. Male SD rats (n=6 per group) were anesthetized (sodium pentobarbital, 60 mg/Kg ip) and their livers removed for perfusion (3 mL/min/g) with Krebs-Henseleit buffer. Normothermic livers (NT) were perfused at 37 °C thoroughly the experiment (30 min). Hypothermic livers were perfused 15 min at 37 °C and then were switched to cold at 26 °C (HT26) or 22 °C (HT22) further 15 min. We measured 4-HNE protein adducts and advanced oxidation protein products (AOPP); the expression of ubiquitin conjugates and subunits of the proteasome 26S: the 19S regulator (Rpt1, Rpt6 and phosphoRpt6 proteasomal ATPases) and the 20S core. The 26S β5 proteasome proteolytic activity was also assayed. Data (mean ± SEM) was analyzed by ANOVA. Our results showed that hypothermic perfusion leads to protein oxidation as demonstrated by increased HNE-protein adducts and AOPP (p<0.05). As regards to the UPS, the presence of poly-ubiquitinated proteins was higher in hypothermia (p<0.001). No differences were observed in the expression of the 20S. The expression of Rpt1 and Rpt6 did not change. However, we found a significant increase in the ratio phospho-Rpt6/Rpt6 from NT (0.59±0.06) to HT26 (1.06±0.09, p<0.05) and HT22 (1.24±0.1, p<0.01). Rpt6 S120 phosphorylation has been suggested to cause an enhancement in the 26S proteasome activity. Accordingly, the 26S β5 activity was significantly increased in HT26 (46%) and HT22 (42%) groups (p<0.01). These results suggest that phosphorylation of 19S subunit, Rpt6 at S120, which is part of the 26S proteasome, activates the proteasome during hypothermia. This is likely to be an important mechanism to quickly increase in the activity to the proteasome independent of increasing the amount of the proteasome. Conclusion: Hypothermic perfusion induces protein oxidation. The UPS is activated to repair this damage, which partly increases its activity due to the Rpt6 subunit phosphorylation.