This study was designed to assess the relationship between the distribution of the monocationic 99mTc-hexakis-2-methoxy, 2-methylpropylisonitrile (99mTc-sestamibi) and regional myocardial blood flow (RMBF) in swine. The left anterior descending coronary artery (LAD) was totally occluded and dipyridamole (0.4 mg/kg) was intravenously infused over 4 minutes. 99mTc-sestamibi and 201Tl were coinjected intravenously and a set of microspheres (15 microns) labelled with 113Sn or 55Nb was injected via a left atrial cannula. Animals were put to death at different times after injection of 99mTc-sestamibi/201Tl and the left ventricle was sectioned for gamma spectroscopy. Regression analysis of regional myocardial distribution of 99mTc-sestamibi or 201Tl versus microsphere-determined RMBF demonstrated a linear relationship with flow up to 2.5 ml/min/gm. The myocardial uptake for both 99mTc-sestamibi and 201Tl at higher flow levels was shown not to follow a linear relationship to microsphere-determined RMBF. The first-pass myocardial extraction fractions (%EF) of 99mTc-sestamibi and 201Tl in dogs were 65.5 +/- 2.5% and 82 +/- 3%, respectively (p less than 0.001), at resting flow. At flow levels above the resting flow, a significant decrease in the first-pass extraction fraction for both tracers was shown. At hyperemic flow levels (two to three times the resting flow), the %EF for both tracers are not significantly different. These data provide a basic validation for the utility of 99mTc-sestamibi as a reliable myocardial perfusion imaging agent.