To clarify the energy-dependency of biliary excretion of a diagnostic dye, the effect of ATP depletion on biliary excretion was investigated. Biliary excretion of indocyanine green (ICG) in rat liver reached a maximum 20 min after intravenous application. The level of ATP in the liver was lowered to 35.4% of the control value about 5 h after administration of DL-ethionine (100 mg/100 g body weight). The rate of bile flow and the amount of ICG excreted in initial 10 min into the bile of ethionine-treated rats were lowered to 67.5% and 61.3% of the control values, respectively. The amount of ICG excreted for 10 min from 30 min to 40 min after application of ICG in ethionine-treated rats was comparable to that of the control. The level of ICG in blood 5 min after application of ICG was raised by 59.2% of the control value. These results suggest that ATP depletion in the liver caused by treatment with ethionine suppresses the initial rate of uptake and biliary excretion of ICG.