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 (approximately 45% VO(2peak)) until exhaustion (approximately 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 alpha AMPK subunits became progressively more phosphorylated on Thr(172) during exercise eliciting a parallel increase in alpha2 but not alpha1 AMPK activity. In contrast, after 1h of exercise, Ser(221) phosphorylation of acetyl-CoA carboxylase-beta (ACCbeta) peaked at 1h of exercise and returned to resting levels at exhaustion. Protein expression of alpha2 AMPK, alpha1 AMPK or ACCbeta 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 ACCbeta Ser(221) phosphorylation during prolonged exercise.