Software packages are widely used for quantification of myocardial perfusion defects. The quantification is used to assist the physician in his/her interpretation of the study. The purpose of this study was to compare the quantification of reversible perfusion defects by three different commercially available software packages. We included 50 consecutive patients who underwent myocardial perfusion single-photon emission tomography (SPET) with a 2-day technetium-99m tetrofosmin protocol. Two experienced technologists processed the studies using the following three software packages: Cedars Quantitative Perfusion SPECT, Emory Cardiac Toolbox and 4D-MSPECT. The same sets of short axis slices were used as input to all three software packages. Myocardial uptake was scored in 20 segments for both the rest and the stress studies. The summed difference score (SDS) was calculated for each patient and the SDS values were classified into: normal (< 4), mildly abnormal (4-8), moderately abnormal (9-13), and severely abnormal (> 13). All three software packages were in agreement that 21 patients had a normal SDS, four patients had a mildly abnormal SDS and one patient had a severely abnormal SDS. In the remaining 24 patients (48%) there was disagreement between the software packages regarding SDS classification. A difference in classification of more than one step between the highest and lowest scores, for example from normal to moderately abnormal or from mildly to severely abnormal, was found in six of these 24 patients. Widely used software packages commonly differ in their quantification of myocardial perfusion defects. The interpreting physician should be aware of these differences when using scoring systems.