Our previous studies on the acute regulation of glucose transport in perfused rat hearts were extended to explore further the mechanism of regulation by anoxia; to test the effects of palmitate, a transport inhibitor; and to compare the translocation of two glucose transporter isoforms (GLUT1 and GLUT4). Following heart perfusions under various conditions, glucose transporters in intracellular membranes were quantitated by reconstitution of transport activity and by Western blotting. Rotenone stimulated glucose uptake and decreased the intracellular contents of glucose transporters. This indicates that it activates glucose transport via net outward translocation, similarly to anoxia. However, two uncouplers of oxidative phosphorylation produced little or no effect. Increased workload (which stimulates glucose transport) reduced the intracellular contents of transporters, while palmitate increased the contents, indicating that these factors cause net translocation from or to the intracellular pool, respectively. Relative changes in GLUT1 were similar to those in GLUT4 for most factors tested. A plot of changes in total intracellular transporter content vs. changes in glucose uptake was roughly linear, with a slope of -0.18. This indicates that translocation accounts for most of the changes in glucose transport, and the basal pool of intracellular transporters is five times as large as the plasma membrane pool.