Hypothermia appears to be useful in the prevention of tissue damage. Recent reports(2) suggest that the use of hypothermic perfusion media (20°C) can improve the preservation of liver grafts. However, hypothermia can also induce reactive oxygen and nitrogen species (RONS)(1). The initial protection against damage from oxidative stress may require 1) activation of the cellular antioxidant system, and 2) rapid removal of oxidized proteins by the ubiquitin-proteasome system (UPS), among other mechanisms. Objective To study the cellular mechanisms induced by hypothermic perfusion in isolated and perfused rat liver (IPRL), focusing on the production of oxidative stress, antioxidant activity, and the UPS system. The procedure was approved by the University of Barcelona’s Committee for Animal Care and followed European Community guidelines. Sprague-Dawley male rats (n=6 per group) were anesthetized with I.P. sodium pentobarbital (60 mg/Kg of b.w.) and their livers were removed for perfusion in a non-recirculating system at a flow rate of 3 mL/ min/g liver with Krebs-Henseleit buffer. Rats were killed by anesthesia overdose. Normothermic livers (NT) were perfused at 37 °C (30 min). Hypothermic livers were perfused at 37° (15 min) and were then switched to cold medium at 26 °C (HT26) or 22 °C (HT22) (15 min). In liver homogenates we determined nitric oxide (NOx), lipid peroxidation (TBARS), modified proteins (4-HNE adducts) and nitrated proteins (nitrotyrosine). As antioxidants, we determined glutathione (GSH) and the activity of the enzymes GSH-peroxidase and GSH-reductase. Expression of the ubiquitin-protein conjugates and the levels of proteasome 26S subunits were also analysed. Data were expressed as means ± standard error and were analysed by two-way ANOVA. Oxidative stress is induced in hypothermic perfusion, as demonstrated by increased levels of nitric oxide (from 1.48±0.14 nmol/mg protein in NT to 2.27±0.17 in HT26), TBARS (from 0.36±0.01 nmol/mg prot in NT to 0.48±0.05 in HT 26) and 4-HNE adducts (a 34% increase). Nitrotyrosine levels were not significantly different. In the HT22 group we found increased levels of GSH and higher activity of the GSH-reductase, which may explain the better turnover in the GSH/GSSG system. As regards the UPS system, poly-ubiquitinated proteins were higher in the HT groups; there were no differences in the expression of the 26S proteasome subunits. Conclusion In the IPRL system, hypothermic perfusion produces oxidative stress, induces the activation of the glutathione antioxidant system and increases ubiquitin-conjugated proteins. A fuller understanding of the cellular mechanisms activated by hypothermic perfusion will help to explore its potential use as a protective strategy for preserving organs.