AMP-activated protein kinase (AMPK) plays a central role in regulating metabolism and energy homeostasis. It achieves its function by sensing fluctuations in the AMP:ATP ratio. AMP deaminase (AMPD) converts AMP into IMP, and the AMPD1 isoenzyme is expressed in skeletal muscles. Here, effects of pharmacological inhibition and genetic deletion of AMPD were examined in contracting skeletal muscles. Pharmacological AMPD inhibition potentiated rises in AMP, AMP:ATP ratio, AMPK Thr172, and acetyl-CoA carboxylase (ACC) Ser218 phosphorylation induced by electrical stimulation, without affecting glucose transport. In incubated extensor digitorum longus and soleus muscles from Ampd1 knockout mice, increases in AMP levels and AMP:ATP ratio by electrical stimulation were potentiated considerably compared with muscles from wild-type mice, whereas enhanced AMPK activation was moderate and only observed in soleus, suggesting control by factors other than changes in adenine nucleotides. AMPD inhibitors could be useful tools for enhancing AMPK activation in cells and tissues during ATP-depletion.