Tetrofosmin is a 99mTc-labeled myocardial perfusion imaging agent that has shown encouraging results in Phase I and II clinical trials. The purpose of this study was to determine the biokinetics of this agent following administration during exercise and at rest in order to determine an optimal imaging protocol. Twenty patients with suspected coronary artery disease underwent symptom-limited treadmill exercise. Six to 8 mCi of 99mTc-tetrofosmin was injected at peak exercise and 22-24 mCi was injected 4 hr later at rest. Serial 5-min planar images were obtained in the left anterior oblique view at 5, 10, 15, 30, 60, 120 and 180 min after the radiotracer injection. Regions of interest were drawn on the serial images around the entire heart and portions of liver, lung, spleen, gallbladder and gastrointestinal tract. Average decay-corrected counts per pixel in each organ were plotted against time. In addition, heart-to-adjacent organ ratios were also determined. On stress images, the heart had the highest activity at all times, with the exception of gallbladder in the first 15 min. On rest images, the gallbladder, liver and gastrointestinal tract initially had higher activity than the heart; but the activity in these organs cleared rapidly over the subsequent 30-60 min. Heart-to-adjacent organ ratios were > 1.0 at all times in the stress images. Heart-to-organ ratios were < 1.0 in the first 15 min on the rest images for the liver and gastrointestinal tract. However, 30 min later, all ratios on the rest images were > or = 1.0. Technetium-99m-tetrofosmin images were considered to be of good to excellent quality with good myocardial delineation and adequate contrast between the heart and background. These These observations indicate that a convenient one-day tetrofosmin imaging protocol similar in duration to conventional 201Tl imaging is feasible.