Gender-matched stress normal limits and criteria for abnormality for rest-stress 99mTc-sestamibi same-day myocardial perfusion imaging were developed and validated in 160 patients who were imaged using previously developed optimized acquisition, processing and quantitative protocols. The gender-matched mean and standard deviation of the normal response were calculated using 35 male and 25 female patients with a < 5% likelihood of coronary artery disease. Receiver-operating curve analysis using expert visual interpretation as the "gold standard" was used to determine the optimal criteria for abnormality detection, in terms of standard deviations from the mean and minimum defect size for each of the four major zones of the polar map, in a pilot population consisting of an additional 35 male and 25 female patients with a variety of perfusion defects. The optimum standard deviations resulted in the following true-positive/true-negative rates when quantitative results were compared to visual analysis for the anterior, septal, lateral, and inferior segments in the combined male and female pilot populations: 84%/86%, 70%/75%, 86%/76% and 69%/76%, respectively. The final criteria were then applied to a prospective population consisting of 33 male and 7 female patients. This analysis resulted in the following true-positive/true-negative rates for overall perfusion abnormalities and abnormalities of the LAD, LCX and RCA vascular territories: 97%/67%, 94%/73%, 73%/90% and 72%/91%, respectively. The optimized 99mTc-sestamibi stress normal limits and criteria for abnormality correlate well with expert visual interpretation of stress myocardial perfusion defects.