Objectives. This study assessed the validity of a novel approach to myocardial perfusion scintigraphy that provides the opportunity to avoid the drawbacks of standard same-day rest/stress technetium-99m sestamibi myocardial perfusion studies by using separate-acquisition dual-isotope rest thallium-201 and exercise technetium-99m sestamibi single-photon emisson computed tomography (SPECT). Background. Standard seme-day rest/stress technetium-99m sestamibi myocardial perfusion studies are cumbersome, associated with a potential decrease in perceived stress defect severity compared with thallium-201 due to the presence of rest technetium-99m sestamibi and may be enable to differentiate hibernating from infarcted myocardium. Methods. The dual-isotope procedure was performed in 63 patients without previous myocardial infarction undergoing coronary angiography to evaluate sensitivity and specificity for coronary artery disease and in 107 patients with a low (<5%) likelihood of coronary artery disease to evaluate normalcy rate. To validate defect reversibility, the dual-isotope SPECT study was compared with stress/rest technetium-99m sestamibi SPECT studies in a separate group of 31 patients with previous documented myocardial infarction who underwent a rest technetium-99m sestamibi study in addition to the dual-isotope SPECT study. Results. In angiographic correlations, dual-isotope SPECT demonstrated high sensitivity for detecting patients with ≥50% stenosis (91 %, 55 patients) and ≥70% stenosis (96%, 52 patients). In a small group of patients, high specificity was also observed (75% for <50% stenosis [8 patients] and 82% for <70% stenosis [11 patients]). A very high normalcy rate of 95% was also found. In the patient group assessed for defect reversibility, in zones with no previous myocardial infarction, segmental agreement for defect type between rest thallium-201 and rest technetium-99m sestamibi studies was 97% (kappa = 0.79, p < 0.001). In myocardial infarct zones, segmental agreement for defect type was 98% (kappa = 0.93, p < 0.001). Image quality was generally good to excellent. Conclusions. Our findings demonstrate that separate-acquisition dual-isotope myocardial perfusion SPECT is accurate for coronary artery disease detection, correlates well with reststress sestamibi studies for assessment of defect reversibility and results in good to excellent image quality. This approach provides an excellent method for the combined assessment of stress myocardial perfusion and myocardial viability.