The relative roles of mitochondrial (mito) ATP-sensitive K+ (mitoKATP) 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 ᠳ mmHg of ventricular pressure (50% preischemia) and released 23 ᠲ IU/g lactate dehydrogenase (LDH). Adenosine (50 卩 during ischemia-reperfusion improved recovery (149 ᠸ mmHg) and reduced LDH efflux (5 ᠱ IU/g). Treatment during ischemia alone was less effective. Treatment with 50 占diazoxide (mitoKATP opener) during ischemia and reperfusion enhanced recovery and was equally effective during ischemia alone. A3 agonism [100 nM 2-chloro-N 6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide], A1 agonism (N 6-cyclohexyladenosine), and AK inhibition (10 占iodotubercidin) all reduced necrosis to the same extent as adenosine, but less effectively reduced contractile dysfunction. These responses were abolished by 100 占5-hydroxydecanoate (5-HD, mitoKATP channel blocker) or 3 占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 mitoKATP 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.