While GLUT1 is thought to be responsible for basal glucose uptake in cardiac myocytes, little is known about its relative distribution between the different plasma membranes and cell types in the heart. GLUT4 translocates to the myocyte surface to increase glucose uptake in response to a number of stimuli. The mechanisms underlying ischemia- and insulin-mediated GLUT4 translocation are known to be different, raising the possibility that the intracellular destinations of GLUT4 following these stimuli also differ. Using immunogold labelling, we describe the cellular localisation of these two transporters, and investigate whether insulin and ischemia induce differential translocation of GLUT4 to different cardiac membranes. Immunogold labelling of GLUT1 and GLUT4 was performed on left-ventricular sections from isolated hearts following 30 minutes of either insulin, ischemia or control perfusion. In control tissue, GLUT1 was predominantly (76%) localised in the capillary endothelial cells, with only 24% of total cardiac GLUT1 present in myocytes. GLUT4 was found predominantly in myocytes, distributed between sarcolemmal and T-tubule membranes (1.84±0.49 and 1.54±0.33 golds.mm-1 respectively), and intracellular vesicles (127±18 golds.mm-2). Insulin increased T-tubule membrane GLUT4 content (2.8±0.4 golds.mm-1, p<0.05), but had less effect on sarcolemmal GLUT4 (1.72±0.53 golds.mm-1). Ischemia induced greater GLUT4 translocation to both membrane types (4.25±0.84 and 4.01±0.27 golds.mm-1 respectively p<0.05). The localisation of GLUT1 suggests a significant role in transporting glucose across the capillary wall prior to myocyte uptake via GLUT1 and GLUT4. We demonstrate independent spatial translocation of GLUT 4 under insulin or ischemic stimulation, and propose independent roles for T-tubular and sarcolemmal GLUT4.