Small animal imaging of cardiovascular disease using single photon emission tomography (SPECT) can be used to provide quantitative measurements of myocardial infarct. The purpose of this study was to demonstrate the accuracy of pinhole SPECT imaging with [99mTc]sestamibi for estimation of infarct size in a rat model of coronary artery disease. Nine rats had their left anterior descending artery ligated to induce a region of myocardial infarct. These animals were injected with 37 MBq [99mTc]sestamibi, and, 1 h later, scanned on a pinhole SPECT system for 30 min. The defect size measured with SPECT, which was dependent on a threshold applied to the short axis circumferential profiles, was compared against the gold standard triphenyltetrazolium chloride (TTC) staining. The size of the perfusion deficit measured using [99mTc]sestamibi SPECT compared very favorably with the TTC staining result, for threshold values in the range 50-70%. The optimum threshold was approximately 70%, giving an excellent correlation (R2=0.89, p<0.001). Estimation of infarct size by [99mTc] sestamibi SPECT yielded an excellent agreement with TTC staining. In conclusion, measurement of myocardial infarct with SPECT can be used to study the rat heart in vivo, and provides a quantitative measure of myocardial viability.