The relationship between the phosphoenergetic state and gluconeogenesis in the liver after ischemic damage was investigated using living rats. The ATP level was determined with in vivo 31P nuclear magnetic resonance spectroscopy, and gluconeogenesis was evaluated with in vivo 31C NMR spectroscopy using L-[3-13C]alanine as a tracer. These two measurements were alternated repeatedly. The rats were divided into three groups: without ischemia (group A); with 10 min ischemia (group B); and with 30 min ischemia (group C). ATP was depleted to 20% of the preischemic state after 10 min ischemia and this level was maintained during 30 min ischemia. After reperfusion, the ATP level was partially restored, but the recovery was smaller in group C. Infusion of [3-13C]alanine was started immediately after the reperfusion. In vivo 13C NMR disclosed changes in the alanine C3, glutamine/glutamate C2 and C3, glucose C1-6, and glycogen C1 signals in the liver. After 60 min infusion of [3-13C]alanine, the ATP level correlated negatively with the signal intensity of alanine (r = -0.664, p = 0.008) and positively with those of glucose and glyogen (r = 0.586, p = 0.023, and r = 0.643, p = 0.011, respectively). These results suggest that the ATP level participates in gluconeogenesis and glycogenesis in the liver. Such multinuclear in vivo NMR observations might uncover new aspects of the metabolic function of the liver in the in vivo state.