A proposed mechanism for irreversible ischemic liver damage has been peroxidation of membrane phospholipids by free radicals. However, the hepatocyte is laden with enzymes which are antioxidants and, therefore, ought to be relatively resistant to oxidative injury. To test the hypothesis that free radical damage from ischemia and reperfusion of the liver is a nonparenchymal cell process, we studied an in vivo model of ischemia. A point of transition from reversible to irreversible ischemia was defined at greater than or equal to 60 min of total ischemia by serial measurements of ATP at control, end of ischemia, and end of reperfusion periods (n = 6 each). Nonparenchymal cells were separated out of 10 livers in each ischemic group using a Percoll gradient. Second derivative spectroscopy did not detect conjugated dienes in any hepatocellular fraction, total cellular, mitochondrial, or microsomal, but did in the nonparenchymal cell fractions of livers from the 60- and 90-min ischemia groups. This in vivo study shows that irreversible ischemia in the rat liver is associated with free radical lipid peroxidation, but that the nonparenchymal cells rather than hepatocytes are the focus of this injury.