Background: Myocardial perfusion single-photon emission computed tomography (MPS) can be used to assess myocardium at risk in occlusive coronary ischaemia. The aim was to develop a method to quantify myocardium at risk as perfusion defect size on ex vivo MPS using co-registration and fusion with ex vivo magnetic resonance imaging (MRI). Methods: Pigs (n = 19) were injected 99mTc-tetrofosmin prior to concluding 40 min of coronary artery occlusion, followed by reperfusion and MRI contrast injection. The excised heart was imaged with T1-weighted MRI and MPS, and images were co-registered using freely available software (Segment v1.8, http://segment.heiberg.se). The left ventricle was semi-automatically delineated in MRI and copied to MPS. The threshold for a MPS perfusion defect was defined as the mean counts in the MPS image at the MRI-determined border between remote myocardium and air. The threshold was measured using count maxima set to the 100th-95th percentile of counts within the myocardium. The count maximum that gave the lowest threshold variability (SD) was considered the most robust. Results: A count maximum using the 100th percentile yielded a threshold of (mean ± SD) 55 ± 6·2%. This method showed the lowest SD compared to 99th-95th percentile count maxima (6·6-7·2%). Conclusions: We describe a method for objective quantification of myocardium at risk as perfusion defect size on MPS using knowledge of the anatomy of the myocardium from co-registered MRI. This enables simultaneous quantification of myocardium at risk by MPS and infarct size by MRI for the evaluation of treatments for myocardial infarction.