Objectives, This study sought to develop a dual-isotope single-acquisition single-photon emission computed tomographic (SPECT) protocol using a multihead SPECT camera equipped with an ultra-high energy collimator to evaluate rest metabolism/stress perfusion simultaneously with fluorine-18 (F18) deoxyglucose/technetium-99m (Tc-99m) 2-hexakis-2-methoxy-2-methylpropyl isonitrile (MIBI). Background. The most accurate and logistic method of identifying injured but viable myocardium remains a diagnostic challenge, Methods, Sixty-five patients were given 25 to 50 g of glucose and, after similar to 60 min, an injection of 370 MBq (10 mCi) of F-18 fluorodeoxyglucose. After a 35-min distribution phase, patients underwent exercise or pharmacologic stress followed by administration of 925 MBq (25 mCi) of Tc-99m MIBI. Five patients underwent F-18 fluorodeoxyglucose positron emission tomography before dual-isotope SPECT. Results. With a window of 20% for both photopeaks and a technelium-99m/fluorine-18 concentration of 3.2:1, the ''spillover'' from fluorine-18 into the technetium-99m window is <6% of the total counts in the window in patients with a normal distribution of both radiopharmaceuticals. Phantom images clearly demonstrated cardiac defects measuring 2 x 1 and 2 x 0.5 cm, There was no significant difference in the images of the five patients who underwent both positron emission tomography and SPECT, Fifty-seven patients (mean [+/-SD] age 55 +/- 15 years, range 25 to 83; 38 men, 19 women) had satisfactory images and were included in the study, Twenty-one patients had normal study results; 15 had mismatched defects; 14 had matched defects; and 7 had both matched and mismatched defects, Twenty-three patients (mean age 54 +/- 6 years, range 30 to 83; 14 men, 9 women) underwent coronary angiography within 3 months of dual-isotope SPECT, There were seven normal studies, eight with mismatched defects, one with a matched defect and seven with matched and mismatched defects, When stenosis >70% was used as the criterion for a diagnosis of coronary artery disease, dual-isotope SPECT had a sensitivity of 100%, specificity of 88%, positive predictive value of 93%, negative predictive value of 100% and an accuracy of 96%. Conclusions. Dual-isotope SPECT may provide an alternative, accurate, cost-effective method to nitrogen-13 ammonia/F-18 fluorodeoxyglucose positron emission tomography or thallium-201 reinjection for identifying injured or dysfunctional but viable myocardium.