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BACKGROUND: The purpose of this study was to determine the relative image quality and interobserver variability among four readers for 201Tl and 99mTc-labeled tetrofosmin myocardial perfusion images. 99mTc-labeled perfusion agents, with near-optimal physical characteristics for gamma camera imaging, may allow for superior image quality and improved consistency of interpretation. However, most studies to date have demonstrated only similarity in the diagnostic accuracy between technetium agents and thallium. Tetrofosmin is a recently developed 99mTc-labeled agent that has shown promising results in early clinical trials. METHODS AND RESULTS: A multicenter, open-label trial was performed during which treadmill exercise thallium and tetrofosmin scintigraphy was performed within a 2-week period of each other in 216 subjects. Image quality was evaluated subjectively and scans were interpreted in a blinded, independent fashion by four readers. Perfusion abnormalities were graded as consistent with ischemia, infarction, or mixed and were described both globally and regionally. Interobserver variability was assessed by use of the kappa statistic, and receiver-operator curves were compared for each observer for the diagnostic accuracy of each agent. More tetrofosmin images were of excellent quality than with thallium (52% vs 28%; p < 0.05), and when differences in quality were noted between the agents, tetrofosmin was more often superior (p < 0.0001). The interobserver variability was lower with tetrofosmin scintigraphy because generally higher kappa values were noted, especially in the lateral wall. Higher receiver-operator curve areas indicative of improved diagnostic accuracy were noted among the four readers for tetrofosmin in 80% of vascular territories. CONCLUSIONS: 99mTc-labeled tetrofosmin scintigraphy yields images of improved quality compared with thallium, and there is an overall improvement in the consistency of image analysis associated with the use of tetrofosmin.