This study was designed to determine the redistribution potential for technetium-99m (<sup>99m</sup>Tc)-hexakis-2-methoxy isobutyl isonitrile (<sup>99m</sup>Tc-Sestamibi) as compared to thallium-201 (<sup>201</sup>T1) in a transiently ischemic swine heart model. The left anterior descending coronary artery (LAD) was totally occluded for 10 min. One minute prior to the release of the LAD, <sup>99m</sup>Tc-Sestamibi, <sup>201</sup>TI and a set of <sup>95</sup>Nb-radiolabeled microspheres (15 µm) were injected. A second set of <sup>51</sup>Cr-radiolabeled microspheres was injected prior to sacrifice in order to document reflow. Animals were sacrificed at different times post-LAD release (ranging from 1 min to 4 h). The left ventricle was sectioned into 0.2- to 0.5-gram pieces for the gamma spectroscopic counting of the <sup>99m</sup>Tc-Sestamibi, <sup>201</sup>TI and radiolabeled microspheres. Linear regression analysis of radiotracer localization versus microsphere-determined regional myocardial blood flow (rMBF) demonstrates an initial slight filling in of <sup>99m</sup>Tc-Sestamibi into transiently ischemic zones, with subsequent stable kinetics up to 4 h (i.e., it does not further redistribute). In comparison, the ischemic to normal ratios for <sup>201</sup>TI activity increase progressively in a time-dependent manner. These differences between<sup>99m</sup> Tc-Sestamibi and <sup>201</sup>TI might be explained on the basis of their blood clearance kinetics and/or their net clearance from normal and ischemic zones of the heart. It is concluded that <sup>99m</sup>Tc-Sestamibi is a stable and reliable indicator for rMBF over time, and that the lack of normalization of <sup>99m</sup>Tc-Sestamibi into transient ischemic zones will necessitate two separate injections for differentiation between ischemia and persistent defects.