Although there is increasing interest in the automatic processing of tomographic radionuclide ventriculography (TRV) studies, validation is mainly limited to a comparison of TRV results with data from planar radionuclide ventriculography (PRV) or gated perfusion single photon emission computed tomography (SPECT). The aim of this study was to use a dynamic physical cardiac phantom to validate the ejection fraction (EF) and volumes from PRV and TRV studies. A new dynamic left ventricular phantom was constructed and used to obtain 21 acquisitions in the planar and tomographic mode. The directly measured volumes and EFs of the phantom during the acquisitions were considered as the gold standard for comparison with TRV and PRV. EFs were calculated from PRV by background-corrected end-diastolic and end-systolic frames. Volumes and EFs were calculated from TRV by region growing with different lower thresholds to search for the optimal threshold. EF from PRV correlated significantly with the real EF (r=0.94, P=0.00). The optimal threshold value for volume calculation from TRV in 336 cases was 50% (r=0.98, P=0.00) yielding the best slope after linear regression. When considering these calculated end-diastolic and end-systolic volumes, EF correlated well (r=0.99, P=0.00) with the real EF, and this correlation was significantly (P=0.04) higher than that of the EF from PRV. Our experiments prove that EF measured by TRV yields more accurate results compared with PRV in dynamic cardiac phantom studies.