Gated myocardial single-photon emission tomography (SPET) allows the evaluation of left ventricular ejection fraction (LVEF), but temporal undersampling may lead to systolic truncation and ejection fraction underestimation. The aim of this study was to evaluate the impact of temporal sampling on thallium gated SPET LVEF measurements. Fifty-five consecutive patients (46 men, mean age 62+/-12 years) with a history of myocardial infarction (anterior 31, inferior 24) were studied. All patients underwent equilibrium radionuclide angiography (ERNA) and gated SPET 4 h after a rest injection of 185 MBq (5 mCi) of thallium-201 using either 8-interval (group 1, n=25) or 16-interval gating (group 2, n=30). In group 2, gated SPET acquisitions were automatically resampled to an 8-interval data set. Projection data were reconstructed using filtered back-projection (Butterworth filter, order 5, cut-off 0.20). LVEF was then calculated using commercially available software (QGS). A higher correlation between gated SPET and ERNA was obtained with 16-interval gating (r=0.94) compared with the resampled data set (r=0.84) and 8-interval gating (r=0.71). Bland-Altman plots showed a dramatic improvement in the agreement between gated SPET and ERNA with 16-interval gating (mean difference: -0.10%+/-5%). Using multiple ANOVA, temporal sampling was the only parameter to influence the difference between the two methods. When using 8-interval gating, gated SPET LVEF was overestimated in women and underestimated in men (ERNA minus gated SPET = -4.0%+/-9.6% in women and 3.6%+/-7.6% in men, P=0.01). In conclusion, 16-interval thallium gated SPET offered the best correlation and agreement with ERNA, and should be preferred to 8-interval gated acquisition for LVEF measurement.