Abstract Background. New algorithms were evaluated for their efficacy in detecting and quantifying serial changes in myocardial perfusion from single photon emission computed tomography (SPECT). Methods and Results. We generated 72 simulations with various left ventricular positions, sizes, count rates, and perfusion defect severities using the nonuniform rational B-splines (NURBs)-based CArdiac Torso (NCAT) phantom. Images were automatically aligned by use of both full linear and rigid transformations and quantified for perfusion by use of the CEqual program. Changes within a given perfusion defect were compared by use of a Student t test before and after registration. Registration approaches were compared by use of receiver operating characteristic analysis. Changes of 5% were not detected well in single patients with or without alignment. Changes of 10% and 15% could be detected with false-positive rates of 15% and 10%, respectively, in single studies if alignment was performed before perfusion analysis. Alignment also reduced the number of studies necessary to demonstrate a significant perfusion change (P < .05) in groups of patients by about half. Conclusion. Comparison of mean uptake by t values in SPECT perfusion defects can be used to detect 10% and greater differences in serial perfusion studies of single patients. Image alignment is necessary to optimize automatic detection of perfusion changes in both single patients and groups of patients. (J Nucl Cardiol 2005;12:302-10.) Key Words: Single photon emission computed tomography • t values • myocardial perfusion