Mechanisms regulating ischemia and reperfusion (I/R)-induced changes in mRNA translation in the heart are poorly defined, as are the factors that initiate these changes. As cellular energy status affects mRNA translation under physiological conditions, it is plausible that I/R-induced changes in translation may in part be a result of altered cellular energy status. Therefore, the purpose of the studies described herein was to compare the effects of I/R with those of altered energy substrate availability on biomarkers of mRNA translation in the heart. Isolated adult rat hearts were perfused with glucose or a combination of glucose plus palmitate, and effects of I/R on various biomarkers of translation were subsequently analyzed. In comparison to hearts perfused with glucose plus palmitate, hearts perfused with glucose alone exhibited increased phosphorylation of eukaryotic elongation factor (eEF)2, the -subunit of eukaryotic initiation factor (eIF)2, and AMP-activated protein kinase (AMPK), and also exhibited enhanced association of eIF4E with eIF4E binding protein (4E-BP)1. Regardless of the energy substrate composition of the buffer, phosphorylation of eEF2 and AMPK were greater than control values following ischemia. Phosphorylation of eIF2 and eIF4E, and the association of eIF4E with 4E-BP1, were also greater than control values following ischemia, but only in hearts perfused with glucose plus palmitate. Reperfusion reversed the ischemia-induced increase in eEF2 phosphorylation in hearts perfused with glucose and reversed ischemia-induced changes in eIF4E, eEF2, and AMPK phosphorylation in hearts perfused with glucose plus palmitate. As many ischemia-induced changes in mRNA translation are mimicked by removal of a metabolic substrate under normal perfusion conditions, the results suggest that cellular energy status represents an important modulator of I/R-induced changes in mRNA translation.