AMP-activated protein kinase (AMPK) acts as a cellular energy sensor. AMPK responds to an increase in AMP concentration ([AMP]) or AMP/ATP. Metformin and phenformin, which are biguanides, have been reported to increase AMPK activity without increasing AMP/ATP. This study tests the hypothesis that these biguanides increase AMPK activity in the heart by increasing cytosolic [AMP]. Groups of isolated rat hearts (n = 5 - 7 each) were perfused with Krebs-Henseleit (KH) buffer with or without 0.2 mM [phenformin] or 10 mM [metformin]. ³¹P NMR measured phosphocreatine, ATP, and intracellular pH were used to calculate cytosolic [AMP]. At various times hearts were freeze clamped and assayed for AMPK activity, the phosphorylation of Thr¹⁷² on the AMPK subunit (AMPK-P) and the phosphorylation of Ser⁷⁹ on ACC, (ACC-P), an AMPK target. In hearts treated with phenformin for 18 min followed by 20 min of KH, [AMP] began to increase at 26 min; at 36 min AMPK activity was elevated. In hearts treated with metformin, [AMP] was increased at 50 min; at 61 min AMPK activity, AMPK-P, and ACC-P were elevated. HPLC measured total AMP content and total AMP/ATP of metformin treated hearts did not increase. In summary, phenformin and metformin both increase AMPK activity and phosphorylation in the isolated heart. The increase in AMPK activity was always preceded by and correlated with increased cytosolic [AMP]. Total AMP content and total AMP/ATP did not change. Cytosolic [AMP] reported metabolically active AMP that triggered increased AMPK activity but measures of total AMP did not.