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In order to evaluate the diagnostic efficacy of the 1-day separate acquisition dual-isotope single-photon emission computed tomography (SPECT) protocol, using 201Tl for the rest and 99mTc-tetrofosmin for the stress images, a consecutive series of patients with suspected or known coronary artery disease (CAD) was studied that also underwent coronary angiography.The results of myocardial SPECT, using a semi-quantitative visual analysis, were acquired in 123 patients and compared with the results of coronary angiography. Sensitivity and specificity were calculated, using thresholds of ≥50 and ≥70% stenosis. As an alternative for specificity, the normalcy rate was determined in a separate group of 87 patients with a <5% pre-test likelihood of CAD.The prevalence of CAD using ≥50 and ≥70% stenosis was 88 and 78%, respectively. The sensitivity for detection of patients with ≥50 and ≥70% stenosis was 94 and 97%, respectively while specificity was 62 and 59%, respectively. The high rate of false positive perfusion defects resulting in a low specificity could be explained by specific clinical issues. However, the routine assessment with additional clinical and electrocardiographic data resulted in a correct interpretation of most of the false positive perfusion defects. The positive predictive value was 92 and 85% and the negative predictive value 46 and 77%, using thresholds of ≥50 and ≥70% stenosis, respectively. The normalcy rate was 91%.The one-day separate acquisition rest 201Tl/stress 99mTc-tetrofosmin SPECT protocol is an efficient procedure for myocardial perfusion scintigraphy with high sensitivity for detection of CAD. Specific clinical issues caused a low value for specificity. Therefore, clinical information and knowledge of the electrocardiogram is essential for a correct interpretation of SPECT images.