Autophagy is a self-degradative process that occurs mainly for house-keeping purposes, such as degradation of long-lived proteins or damaged organelles. Although autophagy is a prominent feature of myocardial ischemia and reperfusion, the functional significance is still unclear and controversial. In this study, we explored effects of the autophagy inhibitor 3-methyladenine (3-MA, 5 mM) in isolated rat left atria subjected to 75 min simulated ischemia-75 min reperfusion (R), when atria were incubated in the presence of 10 mM glucose (G) or G and 1.2 mM palmitate (P), condition observed in most clinical situations of acute myocardial infarction and less well established. ANOVA, n=8. The results showed a significant enhance in LC3-II/LC3-I ratio, an established indicator of autophagy, during R, which was achieved more slowly in the presence of P. These results were accompanied by a decrease of p62, a specific substrate degraded through the autophagy-lysosomal pathway, in both metabolic groups. 3-MA prevented LC3 ratio increase [End stabilization period (ESP)+G:0.94±0.10, ESP+P:1.06±0.02, R+G:1.74±0.16*, R+P:1.68±0.08*, R+G+3-MA:1.00±0.09; R+P+3-MA:0.90±0.07AU; *p<0.05], and p62 clearence in both metabolic conditions (ESP+G:1.14±0.13, ESP+P:1.08±0.03, R+G: 0.63±0.07*, R+P:0.62±0.07*, R+G+3-MA:1.05±0.04, R+P+3-MA:1.10±0.10; *p<0.05). Electron micrographs showed autophagosome like structures during R in the absence of 3-MA. In the presence of autophagy inhibitor a major deterioration of mitochondrial morphology and rate of ATP synthesis was observed at the end of R (R+G:1.1±0.1, R+P:1.2±0.1, R+G+3-MA:0.5±0.1*, R+P+3- MA:0.6±0.1* nmol/min/mg mitochondrial protein; *p<0.05). ATP content recovery was reduced by P and 3-MA (R+G:0.55±0.02, R+P:0.25±0.05*, R+G+3- MA:0.34±0.10*, R+P+3-MA:0.27±0.04* nmol/mg protein; *p<0.05). This results were consistent with the attenuation in contractile reserve (Peak force R+G:91±11, R+P:63±7*, R+G+3-MA:67±8*, R+P+3-MA:65±7* % vs ESP; *p<0.05). Autophagy inhibition, either in the presence of G or P, induced spontaneous tachyarrhythmias (270-300 contractions/min) throughout R, which were attenuated by inner mitochondrial membrane anion channel blocker PK11195 (PK) (R+G:0%, R+P:0%, R+G+3-MA:47%*, R+P+3-MA:57%*, R+G+3-MA+PK:29%#, R+P+3-MA+PK:33%#; *p<0.05 vs R+G, R+P, #p<0.05 all groups), suggesting an involvement of mitochondria in the 3-MA-induced arrhythmias. Finally, as a possible pathway involved in autophagy induction, we explored the effects of AMPK inhibition by Compound C (CC; 10 μM). In both metabolic conditions CC reduced autophagosome like structures as well as LC3 ratio (R+G+CC:1.24±0.05#; R+P+CC:1.28±0.04# AU; #p<0.05 vs ESP+G, ESP+P, R+G, R+P, R+G+3-MA, R+P+3-MA). Present results suggest that autophagy is induced during R, developing cardioprotective effects. P present at high concentrations as an exogenous energy substrate would not exert significant effects on this process in the ischemic-reperfused myocardium.