AMP-activated protein kinase (AMPK) has previously been demonstrated to phosphorylate and inactivate skeletal muscle acetyl-CoA carboxylase (ACC), the enzyme responsible for synthesis of malonyl-CoA, an inhibitor of carnitine palmitoyltransferase 1 and fatty acid oxidation. Contraction-induced activation of AMPK with subsequent phosphorylation/inactivation of ACC has been postulated to be responsible in part for the increase in fatty acid oxidation that occurs in muscle during exercise. These studies were designed to answer the question, "Does phosphorylation of ACC by AMPK make palmitoyl-CoA a more effective inhibitor of ACC?" Purified rat muscle ACC was subjected to phosphorylation by AMPK. Activity was determined on non-phosphorylated and phosphorylated ACC preparations at acetyl-CoA concentrations ranging from 2 to 500 micromolar and at palmitoyl-CoA concentrations ranging from 0 to 100 micromolar. Phosphorylation resulted in a significant decline in the substrate saturation curve at all palmitoyl-CoA concentrations. The Ki for palmitoyl-CoA inhibition of ACC was reduced from 1.7 ± 0.25 µM to 0.85 ± 0.13 µM as a consequence of phosphorylation. At 0.5 mM citrate, ACC activity was reduced to 13% of control values in response to the combination of phosphorylation and 10 micromolar palmitoyl-CoA. Skeletal muscle ACC is more potently inhibited by palmitoyl-CoA after having been phosphorylated by AMPK. This may contribute to low muscle malonyl-CoA values and increasing fatty acid oxidation rates during long-term exercise when plasma fatty acid concentrations are elevated.