The objective of this study was to define the relationship among Kupffer cells, O(2)(-) production, and TNF-alpha expression in the pathophysiology of postischemic liver injury following short and long periods of ischemia. Using different forms of superoxide dismutase with varying circulating half-lives, a monoclonal antibody directed against mouse TNF-alpha, and NADPH oxidase-deficient mice, we found that 45 or 90 min of partial (70%) liver ischemia and 6 h of reperfusion (I/R) produced time-dependent increases in liver injury and TNF-alpha expression in the absence of neutrophil infiltration. Furthermore, we observed that hepatocellular injury induced by short periods of ischemia were not dependent on formation of TNF-alpha but were dependent on Kupffer cells and NADPH oxidase-independent production of O(2)(-). However, liver injury induced by extended periods of ischemia appeared to require the presence of Kupffer cells, NADPH oxidase-derived O(2)(-), and TNF-alpha expression. We conclude that the sources for O(2)(-) formation and the relative importance of TNF-alpha in the pathophysiology of I/R-induced hepatocellular injury differ depending on the duration of ischemia.