Alterations in myocardial glucose metabolism are a key determinant of ischemia-induced depression of left ventricular (LV) mechanical function. As myocardial glycogen is an important source of endogenous glucose, we compared the effects of ischemia on glucose uptake and utilization in isolated working rat hearts in which glycogen content was either replete (G-Replete, 114 µmol.g dry wt^-1) or partially depleted (G-Deplete, 71 µmol.g dry wt^-1). The effects of low-flow ischemia (LFI, 0.5 ml.min^-1) on glucose uptake, glycogen turnover (glycogenolysis and glycogen synthesis), glycolysis, 5'-AMP activated protein kinase (AMPK) activity and GLUT4 translocation were measured. Relative to pre-ischemic values, LFI caused a time-dependent reduction in glycogen content in both G-Replete and G-Deplete groups due to an acceleration of glycogenolysis (by 12-fold and 6-fold, respectively). In G-Replete hearts, LFI (15 min) decreased glucose uptake (by 59%) and did not affect GLUT4 translocation. In G-Depleted hearts, LFI also decreased initially glucose uptake (by 90%) and glycogen synthesis, but after 15 min, when glycogenolysis slowed due to exhaustion of glycogen content, glucose uptake increased (by 31%) in association with a detectable increase in GLUT4 translocation. After 60 min LFI, glucose uptake, glycogenolysis, and glycolysis recovered to near pre-ischemic values in both groups. LFI increased AMPK activity in a time-dependent manner in both groups (by 6-fold and 4-fold, respectively). Thus, when glycogen stores are replete prior to ischemia, ischemia-induced AMPK activation is not sufficient to increase glucose uptake. Under these conditions, an acceleration of glycogen degradation provides sufficient endogenous substrate for glycolysis during ischemia.