During prolonged, low intensity exercise, the type of substrate utilized varies with time. If 5^′AMP-activated protein kinase (AMPK) regulates muscle metabolism during exercise, signaling through AMPK would be expected to change in concordance with changes in substrate utilization. Six healthy, young males cycled (∼45% VO_2peak) until exhaustion (∼3.5h). During exercise, leg glucose uptake and rate of glycogenolysis gradually decreased whereas free fatty acid uptake gradually increased. In the thigh muscle, the α AMPK subunits became progressively more phosphorylated on Thr¹⁷² during exercise eliciting a parallel increase in α2 but not α1 AMPK activity. In contrast, after 1h of exercise, Ser²²¹ phosphorylation of acetyl-CoA carboxylase-β (ACCβ) peaked at 1h of exercise and returned to resting levels at exhaustion. Protein expression of α2 AMPK, α1 AMPK or ACCβ did not change with time. These data suggest that AMPK signaling is not a key regulatory system of muscle substrate combustion during prolonged exercise and that marked activation of AMPK via phosphorylation is not sufficient to maintain an elevated ACCβ Ser ²²¹ phosphorylation during prolonged exercise.