Rasmussen, B. B., and W. W. Winder. Effect of exercise intensity on skeletal muscle malonyl-CoA and acetyl-CoA carboxylase. J. Appl. Physiol. 83(4): 1104–1109, 1997.—Malonyl-CoA is synthesized by acetyl-CoA carboxylase (ACC) and is an inhibitor of fatty acid oxidation. Exercise induces a decline in skeletal muscle malonyl-CoA, which is accompanied by inactivation of ACC and increased activity of AMP-activated protein kinase (AMPK). This study was designed to determine the effect of exercise intensity on the enzyme kinetics of ACC, malonyl-CoA levels, and AMPK activity in skeletal muscle. Male Sprague-Dawley rats were killed (pentobarbital sodium anesthesia) at rest or after 5 min of exercise (10, 20, 30, or 40 m/min at 5% grade). The fast-twitch red and white regions of the quadriceps muscle were excised and frozen in liquid nitrogen. A progressive decrease in red quadriceps ACC maximal velocity (from 28.6 ± 1.5 to 14.3 ± 0.7 nmol ⋅ g⁻¹ ⋅ min⁻¹,P < 0.05), an increase in activation constant for citrate, and a decrease in malonyl-CoA (from 1.9 ± 0.2 to 0.9 ± 0.1 nmol/g,P < 0.05) were seen with the increase in exercise intensity from rest to 40 m/min. AMPK activity increased more than twofold. White quadriceps ACC activity decreased only during intense exercise. We conclude that the extent of ACC inactivation during short-term exercise is dependent on exercise intensity.