Hepatic ischaemia/reperfusion (I/R) injury is a major cause of primary non-function of the graft after liver transplantation. The ability to assess the severity of ischaemic injury would be of prognostic value and allow the possibility of therapeutic interventions. Currently there is no reliable clinical method for assessing the severity of hepatic ischaemic injury. The hepatic handling of Indocyanine Green as a technique for monitoring the severity of I/R injury has been investigated in the present study. A rabbit model of lobar ischaemia was used. At laparotomy, left lobe hepatic ischaemia was produced for 30, 45 or 60 min, followed by 60 min of reperfusion. Liver function tests, bile excretion and flow in the hepatic microcirculation were measured in animals subjected to I/R injury and in controls. Indocyanine Green was given after reperfusion and its concentration was measured directly in the liver using near-infrared spectroscopy. Indocyanine Green hepatic uptake and excretion rates were calculated. I/R injury produced significant increases in hepatic serum enzymes and decreases in bile excretion and hepatic microcirculation in all I/R groups in comparison with controls. There was a significant reduction in Indocyanine Green uptake and excretion in the I/R groups in comparison with controls, which was correlated with the duration of ischaemia. Indocyanine Green uptake was correlated significantly with flow in the hepatic microcirculation, and its excretion was correlated significantly with the severity of liver damage, as reflected by the changes in serum enzymes and bile excretion. In conclusion, I/R injury affects the hepatic handling of Indocyanine Green, and direct quantification of the uptake and excretion of this dye by near-infrared spectroscopy may be used to objectively assess the degree of I/R injury.