AMP-activated protein kinase (AMPK) is activated during muscle contraction in response to the increase in AMP and decrease in phosphocreatine (PCr). Once activated, AMPK has been proposed to phosphorylate a number of targets, resulting in increases in glucose transport, fatty acid oxidation, and gene transcription. Although it has been possible to directly observe phosphorylation of one of these targets, acetyl-CoA carboxylase (ACC) in vitro, it has been more difficult to obtain direct evidence of ACC phosphorylation in contracting skeletal muscle. In these experiments using a phosphoserine antibody to ACC and a phosphothreonine antibody to AMPK, evidence was obtained for phosphorylation and activation of ACC in vitro, in gastrocnemius muscle electrically stimulated at different frequencies, and in muscle from rats running on the treadmill. Significant negative linear correlations between phospho-ACC and ACC activity were observed in all models (P < 0.01). The decline in ACC activity was related to the decrease in PCr and the rise in AMP. A relationship between phospho-AMPK (threonine 172) and activity of AMPK immunoprecipitated with anti-α₂ subunit antibody preparation was also observed. These data provide the first evidence of a direct link between extent of phosphorylation of these proteins at sites recognized by the antibodies and activity of the enzymes in electrically stimulated muscle and in muscle of rats running on the treadmill.