This study examined the results of sequential SPECT dual-isotope imaging with 201Tl and 99mTc-sestamibi in 148 patients, 114 of whom also had coronary angiography and 34 had <5% pretest probability for coronary artery disease (CAD). Methods: Stress thallium/rest sestamibi was used in 82 patients and rest thallium/stress sestamibi in 66 patients. Coronary angiography showed that 17 patients had no CAD, 27 patients had one-vessel CAD, 41 patients had two-vessel CAD and 29 patients had three-vessel CAD. The thallium study (3 mCi) was always done before the sestamibi study (20- 25 mCi). The stress was either symptom-limited treadmill exercise testing or adenosine infusion at a rate of 140 μg/kg/min for 6 min. Results: The study was completed within 2 hr. The stress and rest images were normal in 11 of 17 patients (65%) with no CAD by angiography and in 33 of 34 patients with a low pretest probability of CAD (normalcy rate = 97%). The images were abnormal in 75 patients with CAD (77%). The perfusion pattern was compared to wall motion in 485 segments (97 patients) assessed by contrast ventriculography. There were no or reversible perfusion defects in 357 of 386 segments (92%) with no wall motion abnormality. Conclusion: Sequential dual-isotope imaging is feasible and can be completed in a short period of time and may therefore enhance laboratory throughput and patient convenience.