The 5'-AMP-activated protein kinase (AMPK) is a heterotrimeric complex that functions as an intracellular fuel sensor that affects metabolism, and AMPK is activated in skeletal muscle in response to exercise and energy storage utilization. The diffusibility properties of AMPK α and β isoforms were examined in isolated skeletal muscle fiber segments dissected from rat fast-twitch extensor digitorum longus (EDL) and oxidative soleus muscles that had their surface membrane removed by mechanical dissection. After 1 and 10 min washes, ~60% and 75%, respectively, of the total AMPK pools were found in the diffusible fraction. Following in vitro stimulation that resulted in ~80% decline in maximal force, 20% of the diffusible pool became bound in the fiber. This bound pool was not associated with glycogen, determined by adding a wash step containing amylase. Stimulation of EDL muscles resulted in 28% glycogen utilization and a 40% increase in phosphorylation of the downstream AMPK target, acetyl carboxylase-CoA. This, however, had no effect on the proportion of total β2-AMPK that was phosphorylated in whole muscle homogenates, measured using immunoprecipitation. These findings suggest that in rat skeletal muscle, β2-AMPK is not associated with glycogen and that activation of AMPK by muscle contraction does not dephosphorylate β2-AMPK. These findings question the physiological relevance of the carbohydrate-binding function of β2-AMPK in skeletal muscle.