The present study was designed to investigate the possible participation of Kupffer cells in the development of reperfusion injury of the cold-stored liver graft. In the cold preservation of Kupffer cells with Euro-Collins solution, the proportion of asialo-GM1-positive cells was significantly increased at 12 and 24 hr of storage, and the TNF alpha-producing activity in these cells was approximately fivefold greater than control. Northern blot analysis demonstrated that TNF alpha mRNA was remarkably elevated in the reperfusion of the cold-preserved liver, although that of the prereperfused graft was only slightly induced. The reperfusion experiments of the cold-stored liver graft showed that addition of anti-TNF alpha antibody to the perfusate suppressed the elevation of the effluent levels of GOT and LDH significantly, and that pretreatment with a Kupffer cell inhibitor, gadolinium chloride, inhibited the increase of these enzymes in the effluents almost completely. Histological study revealed deposition of a fibrinlike substance in the sinusoid and the central veins extensively in the reperfused liver graft, whereas no apparent deposition was observed in the gadolinium-pretreated liver. Thus, the present study showed that Kupffer cells were primed by cold preservation with Euro-Collins solution, and then activated when the reperfusion was done. It seems likely that the Kupffer cell activation induced by cold preservation/reperfusion plays a major role in reperfusion injury with sinusoidal microcirculatory disturbance, and that TNF alpha is responsible for the impairment of the reperfused liver graft.