Separate-acquisition rest thallium-201/exercise technetium-99m sestamibi (sestamibi) dual-isotope single-photon emission computed tomography (SPECT) has been shown to be effective for assessment of myocardial perfusion and viability. The present study was designed to validate the dual-isotope approach when used in conjunction with pharmacologic stress. All patients had rest 201TI SPECT followed immediately by adenosine (n = 82) or dipyridamole (n = 50) infusion and sestamibi injection. Sestamibi SPECT was performed 1 hour later. The entire study lasted <2.5 hours. The patient population was categorized into three groups: 51 consecutive patients with coronary angiography and no previous myocardial infarction (group I), 58 consecutive patients with a low prescintigraphic test likelihood of coronary artery disease (group II), and 23 consecutive catheterized patients with remote Q-wave myocardial infarction (group III). For group I patients, the sensitivity and specificity for dual-isotope SPECT were 92% (35 of 38) and 85% (11 of 13), respectively, when ≥50% coronary artery narrowing was considered significant and were 97% (34 of 35) and 81% (13 of 18) respectively, when ≥70% narrowing was considered significant. The normalcy rate among the 58 patients of group II was 96%. Comparisons for pattern of stress-defect reversibility demonstrated that of the 97 stress defects within the infarct zones (group III), 15% were reversible and 85% were nonreversible. In contrast, of the 227 stress defects within the diseased (≥50% stenosis) vessel zones of the group I patients, 93% were reversible and 7% were nonreversible (p < 0.001 vs group III). In conclusion, separate acquisition rest 201-TI/pharmacologic stress sestamibi dual-isotope SPECT is an efficient myocardial perfusion imaging protocol with high accuracy for detection and assessment of angiographically significant coronary artery disease.