Gated SPECT recorded with 16 intervals determines left ventricular (LV) ejection fraction more accurately than does gated SPECT recorded with 8 intervals but produces higher image noise. This study aimed to assess the results from sestamibi and (TI)-T-201 16-interval gated SPECT when both signal-to-noise ratio and spatial resolution were enhanced with an original method of reconstruction. Methods: Forty patients with coronary artery disease underwent (TI)-T-201 and sestamibi 16-interval gated SPECT and, to be used as a reference, cardiac MRI. Assessments of global and regional LV function provided by ordered-subsets expectation maximization (OSEM) with depth-dependant resolution recovery and temporal Fourier filtering were compared with those from conventional filtered backprojection (FBP) previously optimized by screening various filter frequencies and various temporal smoothing levels. Results: For both tracers, LV ejection fraction was determined best when the association of OSEM with depth-dependant resolution recovery was used alone, with temporal Fourier filtering, or with a slight 2-frame temporal smoothing: Mean absolute values of relative errors ranged from 3.2% to 3.6% (4.0%-7.9% for FBP), and coefficient correlation ranged from 0.91 to 0.93 (0.70-0.91 for FBP). Among these 3 reconstruction methods, the association of OSEM with depth-dependant resolution recovery with temporal Fourier filtering provided the highest signal-to-noise ratio, with mean increases of 54% for sestamibi and 80% for (TI)-T-201 when compared with FBP, and the best analysis of segmental contractility, with exact agreement rates with MRI being 73% for (TI)-T-201 and 79% for sestamibi. Conclusion: OSEM associated with temporal Fourier filtering and depth-dependant resolution recovery filtering enhances the LV function assessment provided by sestamibi and (TI)-T-201 16-interval gated SPECT and dramatically reduces image noise, a property that enhances and facilitates image interpretation.