AMP-activated protein kinase (AMPK) is emerging as an important signaling protein during myocardial ischemia. AMPK is a heterotrimeric complex containing an α catalytic subunit and β and γ regulatory subunits. Phosphorylation of Thr 172 in the activation loop of the α subunit by upstream AMPK kinase(s) (AMPKK) is a critical determinant of AMPK activity. However, the mechanisms regulating AMPK phosphorylation in the ischemic heart remain uncertain and were therefore investigated. In the isolated working rat heart, low-flow ischemia rapidly activated AMPKK activity when measured using recombinant AMPK (rAMPK) as substrate. The addition of AMP (10 to 200 μmol/L) augmented the ability of heterotrimeric α 1 β 1 γ 1 or α 2 β 1 γ 1 rAMPK to be phosphorylated by heart AMPKK in vitro, whereas physiologic concentrations of ATP inhibited rAMPK phosphorylation. However, neither AMP nor ATP directly influenced AMPKK activity: they had no effect on AMPKK-mediated phosphorylation of rAMPK substrates lacking normal AMP-binding γ subunits (isolated truncated α 1 1–312 or α 1 β 1 γ 1 rAMPK containing an R70Q mutation in the γ 1 AMP-binding site). Regional ischemia in vivo also increased AMPKK activity and AMPK phosphorylation in the rat heart. AMPK phosphorylation could also be induced in vivo without activating AMPKK: AICAR infusion increased AMPK phosphorylation without activating AMPKK; however, the AMP-mimetic AICAR metabolite ZMP enhanced the ability of heterotrimeric rAMPK to be phosphorylated by AMPKK. Thus, heart AMPKK activity is increased by ischemia and its ability to phosphorylate AMPK is highly modulated by the interaction of AMP and ATP with the heterotrimeric AMPK complex, indicating that dual mechanisms regulate AMPKK action in the ischemic heart.