The effect of exercise intensity on skeletal muscle AMP-activated protein kinase (AMPK) signaling and substrate metabolism was examined in eight men cycling for 20 min at each of three sequential intensities: low (40 ± 2% Vo₂ peak), medium (59 ± 1% Vo₂ peak), and high (79 ± 1% Vo₂ peak). Muscle free AMP/ATP ratio only increased at the two higher exercise intensities (P < 0.05). AMPK α1 (1.5-fold) and AMPK α2 (5-fold) activities increased from low to medium intensity, with AMPK α2 activity increasing further from medium to high intensity. The upstream AMPK kinase activity was substantial at rest and only increased 50% with exercise, indicating that, initially, signaling through AMPK did not require AMPK kinase posttranslational modification. Acetyl-CoA carboxylase (ACC)-β phosphorylation was sensitive to exercise, increasing threefold from rest to low intensity, whereas neuronal NO synthase (nNOS)μ phosphorylation was only observed at the higher exercise intensities. Glucose disappearance (tracer) did not increase from rest to low intensity, but increased sequentially from low to medium to high intensity. Calculated fat oxidation increased from rest to low intensity in parallel with ACCβ phosphorylation, then declined during high intensity. These results indicate that ACCβ phosphorylation is especially sensitive to exercise and tightly coupled to AMPK signaling and that AMPK activation does not depend on AMPK kinase activation during exercise.