This study was done to determine whether [99mTc]methoxyisobutyl isonitrile (RP30), a nonredistributing myocardial perfusion agent, could be used to quantify regional myocardial blood flow distribution during ischemia and reperfusion, employing sequential injections of tracer, tomographic imaging, and appropriate image subtraction. Dogs underwent transient (6 min) coronary artery occlusion, followed by two paired injections of RP30 and radioactive microspheres combined with tomographic imaging, the first during coronary occlusion and the second after 60 min of reperfusion. To obtain a true image representative of reperfusion, the first set of images was corrected for 99mTc decay and subtracted from the second. During occlusion, tissue microsphere content and scintigraphic RP30 activity in the center of the ischemic region (both expressed as a fraction of the nonischemic region) were closely correlated, although RP30 consistently exceeded microsphere content (0.43 +/- 0.03 vs. 0.24 +/- 0.04, p less than 0.01). Direct tissue counting of RP30 confirmed its relative excess in ischemic myocardium. Reperfusion was successful in 7/8 dogs, with an increase in RP30 activity to 0.98 +/- 0.04 compared to 0.89 +/- 0.03 for microspheres (p = N.S.). In one dog with microsphere-documented persistent ischemia, the RP30 defect was still present after reflow. Our results indicate that because of the lack of myocardial clearance and redistribution, repeat injections of RP30 can be used to quantify serial changes in regional myocardial blood flow.