AMP-activated protein kinase (AMPK) is considered a major regulator of skeletal muscle metabolism during exercise. Indeed, AMPK is activated during exercise and activation of AMPK by 5-aminoimidazole-4-carboxyamide-ribonucleoside (AICAR) increases skeletal muscle glucose uptake and fat oxidation. However, we have previously shown that although AMPK activity increases 8-10 fold during ∼120 min of exercise at ∼65% VO2 peak in untrained individuals, there is no increase in these individuals after only 10 days of exercise training (longitudinal study). In a cross-sectional study we examined whether there is also a lack of activation of skeletal muscle AMPK during 120 min of cycling exercise at 65% VO2 peak in endurance-trained individuals. Eleven untrained (UT; VO2 peak = 37.9 ± 1.8 ml.kg-1 .min-1 ) and seven endurance trained (ET; VO2 peak = 61.8 ± 0.9 ml.kg-1 .min-1 ) males completed 120 min of cycling exercise at 66 ± 1% VO2 peak (UT: 100 ± 7 watts; ET: 190 ± 6 watts). Muscle biopsies were obtained at rest and following 30 and 120 min of exercise. Muscle glycogen was significantly (P < 0.05) higher before exercise in ET and decreased similarly during exercise in the ET and UT individuals. Exercise significantly increased calculated skeletal muscle free AMP content and more so in the UT individuals. Exercise significantly (P < 0.05) increased skeletal muscle AMPK α2 activity (7-fold), AMPK α Thr172 phosphorylation (2-fold) and ACCβ Ser222 phosphorylation (2-fold) in the UT individuals but not in the ET individuals. These findings indicate that AMPK is not an important regulator of exercise metabolism during 120 min of exercise at 65% VO2 peak in endurance trained men. This article is protected by copyright. All rights reserved.