Objective: Myocardial glucose transport is enhanced by hormonal and other stimuli such as ischemia and hypoxia which induce glucose transporter 4 (GLUT4) translocation. Whether insulin and ischemia share a common signaling mechanism is not yet known. This study investigated whether phosphatidylinositol 3-kinase (PI3K), a signaling intermediate of the insulin-responsible pathway, also participates in the ischemia-induced stimulation of glucose. Methods: Isolated Langendorff-perfused Sprague-Dawley rat hearts were subjected to 100 nmol/l insulin or 15 min of no-flow ischemia with/without 1 μmol/l wortmannin, an inhibitor of PI3K. After perfusion, relative subcellular glucose transporter GLUT4 distribution was assessed by membrane fractionation and immunoblotting and compared to controls. Uptake kinetics of the glucose analog [¹⁸F]fluoro-deoxyglucose (FDG) were also studied during perfusion of rat hearts. Results: GLUT4 translocation to the plasma membrane (PM) was increased by insulin 1.8-fold and by ischemia 2.4-fold (P<0.05). FDG uptake was increased by insulin 6.0-fold and by ischemia 6.2-fold (P<0.05). Wortmannin 1 μmol/l inhibited insulin-mediated translocation of GLUT4 and increase in FDG uptake completely. However, it did not show any effect on ischemia-stimulated GLUT4 translocation or on ischemia-induced increase in FDG utilization. A significant correlation was found between relative GLUT4 translocation and FDG uptake in hearts of the insulin series (r = 0.9, P<0.05) and of the ischemia series (r = 0.8, P<0.05). Conclusions: Our results demonstrate that wortmannin did not inhibit ischemia-induced stimulation of myocardial glucose transport, supporting the hypothesis of different signaling pathways for ischemia and insulin.