The purpose of this study was to evaluate the role of Kupffer cell activation in the pathogenesis of reperfusion injury. In a blood-free liver perfusion model, pericentral hypoxia and reperfusion injury occurred. Lactate dehydrogenase (LDH) and malondialdehyde (MDA) release, oxygen uptake, and trypan blue staining were assessed. Within the first 10 min of reflow, LDH and MDA release reached maximal values of 44 U.g-1.h-1 and 115 nmol.g-1.h-1, respectively. Trypan blue cell staining was confined to pericentral regions of the liver lobule. When Kupffer cells were inactivated with GdCl3, release of enzymes and MDA was reduced significantly by > 50%, and hepatic cell death was almost completely absent. Since increases in MDA suggested involvement of free radicals, livers were perfused with phenyl N-t-butylnitrone (5 mM), a spin-trapping agent. Analysis of liver tissue by electron paramagnetic resonance spectroscopy revealed a typical six-line spectrum, providing direct evidence that carbon-centered radicals were generated on reflow. GdCl3 treatment decreased radical adduct formation by approximately 50%. Collectively, these results strongly support the hypothesis that activation of Kupffer cells plays an important role in the pathogenesis of hepatic reperfusion injury.