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BACKGROUND: With multihead gamma cameras both 180- and 360-degree acquisitions of myocardial perfusion are feasible. However, with 99mTC-labeled sestamibi (99mTC-sestamibi) the optimal clinically relevant demonstration of the superiority of 180- versus 360-degree data acquisition has not been performed. METHODS: Seventy-two consecutive patients undergoing 99mTC-sestamibi imaging at rest and stress who had coronary angiography performed within 3 months were enrolled. The results of blinded interpretation of 13 segments per patient for the 180- and 360-degree data were compared for interobserver variability. Sensitivity and specificity of defect localization in the left anterior descending, right coronary, and left circumflex territories for detection of 50% or greater or 70% or greater stenoses by coronary angiography were compared. RESULTS: There was significant segmental agreement of the stress perfusion images between observers for 180-degree (Kappa = 0.63) and 360-degree data (Kappa = 0.58), but the agreement was significantly higher for 180-degree data (p < 0.05). Overall sensitivity for the detection of coronary artery disease as a 50% or greater stenosis in 62 patients was 79% with 180-degree acquisition and 77% with 360-degree acquisition. The specificity for absence of coronary artery disease in 10 patients was also similar at 70% and 80%, respectively. There was no overall difference in detection of individual stenoses with a sensitivity of 54% with 180-degree acquisition and 50% with 360-degree acquisition. Specificity was also similar at 78% and 81%, respectively. CONCLUSION: There is no difference in clinically relevant detection of overall coronary artery disease or individual stenoses using 180- or 360-degree acquisition of 99mTC-sestamibi myocardial perfusion images. However, 180-degree acquisition has superior interobserver reproducibility.