Oxidative stress to vascular endothelium plays an important role in cold ischemia-reperfusion (CIR) injury. We compared mitochondrial and plasma membrane integrity in human endothelial cells after 20-min exposure to 500 microM H2O or 8-hr cold ischemia and simulated reperfusion. In both groups, plasma membrane integrity was maintained but respiration was significantly decreased, as measured by high-resolution respirometry. Uncoupling was more pronounced after H2O exposure compared with CIR. After H2O exposure, complex I respiration was significantly reduced, whereas CIR resulted additionally in a significant inhibition of complex II and IV respiration. Our results point to a partial overlap of the patterns of mitochondrial defects after H2O-mediated and CIR injury. In this respect, H2O exposure proved to be a useful model to study the mechanisms of CIR injury to human endothelial cells, whereas the full pattern of CIR injury could not be induced by a pulse of hydrogen peroxide exposure.