The effect of AMP-activated protein kinase (AMPK) activation on skeletal muscle glucose metabolism was examined in awake rats by infusing them with 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR; 40 mg/kg bolus and 7.5 mg ⋅ kg⁻¹ ⋅ min⁻¹constant infusion) along with a variable infusion of glucose (49.1 ± 2.4 μmol ⋅ kg⁻¹ ⋅ min⁻¹) to maintain euglycemia. Activation of AMPK by AICAR caused 2-deoxy-d-[1,2-³H]glucose (2-DG) uptake to increase more than twofold in the soleus and the lateral and medial gastrocnemius compared with saline infusion and occurred without phosphatidylinositol 3-kinase activation. Glucose uptake was also assessed in vitro by use of the epitrochlearis muscle incubated either with AICAR (0.5 mM) or insulin (20 mU/ml) or both in the presence or absence of wortmannin (1.0 μM). AICAR and insulin increased muscle 2-DG uptake rates by ∼2- and 2.7-fold, respectively, compared with basal rates. Combining AICAR and insulin led to a fully additive effect on muscle glucose transport activity. Wortmannin inhibited insulin-stimulated glucose uptake. However, neither wortmannin nor 8-(p-sulfophenyl)-theophylline (10 μM), an adenosine receptor antagonist, inhibited the AICAR-induced activation of glucose uptake. Electrical stimulation led to an about threefold increase in glucose uptake over basal rates, whereas no additive effect was found when AICAR and contractions were combined. In conclusion, the activation of AMPK by AICAR increases skeletal muscle glucose transport activity both in vivo and in vitro. This cellular pathway may play an important role in exercise-induced increase in glucose transport activity.