Lethal reperfusion injury to sinusoidal endothelial cells occurs after cold ischemic storage of livers and may be responsible for liver graft failure from storage injury. Here, we evaluated potential mechanisms underlying this reperfusion injury. In rat livers stored in Euro-Collins solution for 24 h and reperfused with Krebs-Henseleit bicarbonate buffer, nonparenchymal cell killing showed periportal predominance as assessed by nuclear staining with trypan blue. In livers reperfused in the retrograde direction, the lobular distribution of cell killing was reversed, indicating that cell killing was more rapid in oxygen-rich upstream regions. However, antioxidants, including allopurinol, desferrioxamine, catalase, superoxide dismutase, superoxide dismutase plus catalase, and U74006F, did not reduce cell killing. Similarly, reperfusion with anoxic buffer did not prevent lethal injury. Antioxidants and anoxic reperfusion also did not improve cell viability in livers stored in UW solution. Nevertheless, superoxide generation, as identified by formazan formation from nitroblue tetrazolium, was increased in Kupffer cells after lives storage and reperfusion as compared to unstored livers. Acidification of the reperfusion buffer from pH 7.4 to pH 7.15 reduced overall nonparenchymal cell killing from about 40% to 10%. Moreover, a pH gradient developed across the liver lobule during reperfusion with the effluent 0.2-0.4 pH units more acidic than the influent. This intralobular pH gradient appears to account for the relative sparing of cells in more acidic downstream regions of the lobule. Lower temperatures of reperfusion also reduced lethal injury. In conclusion, Kupffer cells generated superoxide after perfusion of stored rat livers, but formation of oxygen free radicals did not appear to contribute to lethal reperfusion injury to endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)