Background. Previous studies have shown that gated single photon emission computed tomography (SPECT) technetium 99-labeled sestamibi imaging provides accurate and reproducible measurement of left ventricular (LV) ejection fraction (EF), wall motion, and thickening. This study examined the reliability of gated SPECT sestamibi imaging in measuring LV enddiastolic volume (EDV), end-systolic volume (ESV), and stroke volume (SV). Methods and Results. Gated SPECT measurements were compared with an independent nongeometric method based on thermodilution SV and first-pass radionuclide angiographic EF (using a multicrystal gamma camera). Twenty-four patients aged 58 ± 11 years underwent cardiac catheterization and coronary angiography for evaluation of chest pain syndromes. None had primary valvular disease, intracardiac shunts, or atrial fibrillation. Results. The correlation between the two methods were as follows: EDV: r = 0.89, P < .001; ESV: r = .938, P < .001; SV: r = 0.577, P < .001. Bland-Altman plots showed mean differences (± standard deviation [SD]) for EDV of -14.3 ± 33.3 mL, for ESV of -0.4 ± 23.7 mL, and for SV of -13.9 ± 15.2 mL. The reproducibility of measuring EDV and ESV by gated SPECT was very high (r = 0.99 each). Conclusion. Gated 99mTc-labeled sestamibi SPECT provides reproducible LV volume measurements. With validation of volume measurement, gated SPECT provides comprehensive assessment of regional and global LV function. This information is important in many patient groups such as those with ischemic cardiomyopathy, concomitant coronary and valve disease, and those who have had myocardial infarction. It will also be useful to assess the incremental value of LV volumes in risk assessment.