The objective of this study was to define the relationship among Kupffer cells, Oproduction, and TNF-α 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-α, 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-α 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-α but were dependent on Kupffer cells and NADPH oxidase-independent production of O. However, liver injury induced by extended periods of ischemia appeared to require the presence of Kupffer cells, NADPH oxidase-derived O, and TNF-α expression. We conclude that the sources for O formation and the relative importance of TNF-α in the pathophysiology of I/R-induced hepatocellular injury differ depending on the duration of ischemia.