The relative roles of mitochondrial (mito) ATP-sensitive K⁺ (mitoK_ATP) channels, protein kinase C (PKC), and adenosine kinase (AK) in adenosine-mediated protection were assessed in Langendorff-perfused mouse hearts subjected to 20-min ischemia and 45-min reperfusion. Control hearts recovered 72 ± 3 mmHg of ventricular pressure (50% preischemia) and released 23 ± 2 IU/g lactate dehydrogenase (LDH). Adenosine (50 µM) during ischemia-reperfusion improved recovery (149 ± 8 mmHg) and reduced LDH efflux (5 ± 1 IU/g). Treatment during ischemia alone was less effective. Treatment with 50 µM diazoxide (mitoK_ATP opener) during ischemia and reperfusion enhanced recovery and was equally effective during ischemia alone. A₃ agonism [100 nM 2-chloro-N⁶-(3-iodobenzyl)-adenosine-5'-N-methyluronamide], A₁ agonism (N⁶-cyclohexyladenosine), and AK inhibition (10 µM iodotubercidin) all reduced necrosis to the same extent as adenosine, but less effectively reduced contractile dysfunction. These responses were abolished by 100 µM 5-hydroxydecanoate (5-HD, mitoK_ATP channel blocker) or 3 µM chelerythrine (PKC inhibitor). However, the protective effects of adenosine during ischemia-reperfusion were resistant to 5-HD and chelerythrine and only abolished when inhibitors were coinfused with iodotubercidin. Data indicate adenosine-mediated protection via A₁/A₃ adenosine receptors is mitoK_ATP channel and PKC dependent, with evidence for a downstream location of PKC. Adenosine provides additional and substantial protection via phosphorylation to 5'-AMP, primarily during reperfusion.