We have shown that the cellular process of macroautophagy plays a protective role in HL-1 cardiomyocytes subjected to simulated ischemia/reperfusion (sI/R) (Hamacher-Brady et al. in J Biol Chem 281(40):29776-29787). Since the nucleoside adenosine has been shown to mimic both early and late phase ischemic preconditioning, a potent cardioprotective phenomenon, the purpose of this study was to determine the effect of adenosine on autophagosome formation. Autophagy is a highly regulated intracellular degradation process by which cells remove cytosolic long-lived proteins and damaged organelles, and can be monitored by imaging the incorporation of microtubule-associated light chain 3 (LC3) fused to a fluorescent protein (GFP or mCherry) into nascent autophagosomes. We investigated the effect of adenosine receptor agonists on autophagy and cell survival following sI/R in GFP-LC3 infected HL-1 cells and neonatal rat cardiomyocytes. The A(1) adenosine receptor agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA) (100 nM) caused an increase in the number of autophagosomes within 10 min of treatment; the effect persisted for at least 300 min. A significant inhibition of autophagy and loss of protection against sI/R measured by release of lactate dehydrogenase (LDH), was demonstrated in CCPA-pretreated cells treated with an A(1) receptor antagonist, a phospholipase C inhibitor, or an intracellular Ca(+2) chelator. To determine whether autophagy was required for the protective effect of CCPA, autophagy was blocked with a dominant negative inhibitor (Atg5(K130R)) delivered by transient transfection (in HL-1 cells) or protein transduction (in adult rat cardiomyocytes). CCPA attenuated LDH release after sI/R, but protection was lost when autophagy was blocked. To assess autophagy in vivo, transgenic mice expressing the red fluorescent autophagy marker mCherry-LC3 under the control of the alpha myosin heavy chain promoter were treated with CCPA 1 mg/kg i.p. Fluorescence microscopy of cryosections taken from the left ventricle 30 min after CCPA injection revealed a large increase in the number of mCherry-LC3-labeled structures, indicating the induction of autophagy by CCPA in vivo. Taken together, these results indicate that autophagy plays an important role in mediating the cardioprotective effects conferred by adenosine pretreatment.