The purposes of this study were to characterize the performance of a 3-dimensional (3D) ordered-subset expectation maximization (OSEM) algorithm in the quantification of left ventricular (LV) function with 99m Tc-labeled agent gated SPECT (G-SPECT), the QGS program, and a beating-heart phantom and to optimize the reconstruction parameters for clinical applications. Methods: A G-SPECT image of a dynamic heart phantom simulating the beating left ventricle was acquired. The exact volumes of the phantom were known and were as follows: end-diastolic volume (EDV) of 112 mL, end-systolic volume (ESV) of 37 mL, and stroke volume (SV) of 75 mL; these volumes produced an LV ejection fraction (LVEF) of 67%. Tomographic reconstructions were obtained after 10-20 iterations (I) with 4, 8, and 16 subsets (S) at full width at half maximum (FWHM) gaussian postprocessing filter cutoff values of 8-15 mm. The QGS program was used for quantitative measurements. Results: Measured values ranged from 72 to 92 mL for EDV, from 18 to 32 mL for ESV, and from 54 to 63 mL for SV, and the calculated LVEF ranged from 65% to 76%. Overall, the combination of 10 I, 8 S, and a cutoff filter value of 10 mm produced the most accurate results. The plot of the measures with respect to the expectation maximization-equivalent iterations (I · S product) revealed a bell-shaped curve for the LV volumes and a reverse distribution for the LVEF, with the best results in the intermediate range. In particular, FWHM cutoff values exceeding 10 mm affected the estimation of the LV volumes. Conclusion: The QGS program is able to correctly calculate the LVEF when used in association with an optimized 3D OSEM algorithm (8 S, 10 I, and FWHM of 10 mm) but underestimates the LV volumes. However, various combinations of technical parameters, including a limited range of I and S (80-160 expectation maximization-equivalent iterations) and low cutoff values (#10 mm) for the gaussian postprocessing filter, produced results with similar accuracies and without clinically relevant differences in the LV volumes and the estimated LVEF. It erative reconstruction algorithms were recently proposed as a new potential tool for increasing the quality of tomographic images in nuclear medicine. Protocols based on ordered-subset expectation maximization (OSEM) have become the dominant iterative reconstruction procedures in SPECT and PET. The performance of OSEM was compared with that of filtered backprojection (FBP) in several experimental and clinical studies (1-5); OSEM was shown to have better noise properties and higher image contrast and resolution. On the other hand, even though iterative protocols were suggested for cardiac SPECT in the recent European Association of Nuclear Medicine/European Society of Cardiology guidelines (6), controversial results were reported when myocardial perfusion and left ventricular (LV) function were assessed by different iterative methods (maximumlikelihood expectation maximization, iterative-weighted, and 2-dimensional OSEM) (7-16). Standardization ...