A portal venous 5-aminoimidazole-4-carboxamide-1--D-ribofuranoside infusion that results in hepatic 5-aminoimidazole-4-carboxamide-1--D-ribosyl-5-monophospate (ZMP) concentrations of ~4 µmol/g liver potently increases hepatic glycogen breakdown and glucose output. ZMP is an adenosine 5'-monophosphate (AMP) analogue, which mimics the regulatory actions of this nucleotide. The aim of this study was to measure hepatic adenine nucleotide concentrations in response to increasing energy requirements to test the hypothesis that AMP achieves concentrations during exercise consistent with a role in stimulation of hepatic glycogen breakdown and glucose output. Male C57BL/6J mice (27.4 ± 0.4 g) were either: subjected to 35 min of rest (SED, n=8), underwent short-term (35 min) moderate (20 m/min, 5% grade) exercise (ST, n=8), or underwent treadmill exercise at the same work intensity, but until exhaustion (EXH, n=8). Hepatic AMP concentrations were 0.82 ± 0.05, 1.17 ± 0.11, and 2.52 ± 0.16 µmol/g tissue in SED, ST, and EXH, respectively (p<0.05 between all groups). Gastrocnemius AMP concentrations were 0.04 ± 0.01, 0.08 ± 0.01, and 0.11 ± 0.02 µmol/g tissue in SED, ST, and EXH, respectively (p<0.05 between all groups, except ST vs. EXH). Liver energy charge (inversely related to metabolic stress) was 0.66 ± 0.01, 0.58 ± 0.02, and 0.33 ± 0.22 in SED, ST, and EXH, respectively (p<0.05 between all groups). Gastrocnemius energy charge was 0.94 ± 0.01, 0.92 ± 0.01, and 0.91 ± 0.01 in SED, ST, and EXH, respectively. Liver glycogen was 11.6 ± 1.0, 8.8 ± 2.2, and 0.0 ± 0.1 mg/g liver in SED, ST, and EXH, respectively (p<0.05 between all groups). Hepatic AMP-activated protein kinase (AMPK Thr¹⁷²) phosphorylation was 1.00 ± 0.14, 1.96 ± 0.16, and 7.44 ± 0.63 (arbitrary units) in SED, ST, and EXH, respectively (p<0.05 between all groups). Thus, exercise increases hepatic AMP concentrations. Energy charge decreases in liver, but is unaffected in working muscle. These data suggest that the liver is highly sensitive to metabolic demands as evidenced by dramatic changes in cellular energy indicators (AMP) and sensors thereof (AMPK). In conclusion, AMP is sensitively regulated, consistent with it having an important role in hepatic metabolic control.