PURPOSE: Combining the functional information of SPECT myocardial perfusion imaging (SPECT-MPI) and the morphological information of coronary CT angiography (CTA) may allow easier evaluation of the spatial relationship between coronary stenoses and perfusion defects. The aim of the present study was the validation of a novel software solution for three-dimensional (3D) image fusion of SPECT-MPI and CTA. METHODS: SPECT-MPI with adenosine stress/rest 99mTc-tetrofosmin was fused with 64-slice CTA in 15 consecutive patients with a single perfusion defect and a single significant coronary artery stenosis (>or=50% diameter stenosis). 3D fused SPECT/CT images were analysed by two independent observers with regard to superposition of the stenosed vessel onto the myocardial perfusion defect. Interobserver variability was assessed by recording the X, Y, Z coordinates for the origin of the stenosed coronary artery and the centre of the perfusion defect and measuring the distance between the two landmarks. RESULTS: SPECT-MPI revealed a fixed defect in seven patients, a reversible defect in five patients and a mixed defect in three patients and CTA documented a significant stenosis in the respective subtending coronary artery. 3D fused SPECT/CT images showed a match of coronary lesion and perfusion defect in each patient and the fusion process took less than 15 min. Interobserver variability was excellent for landmark detection (r = 1.00 and r = 0.99, p < 0.0001) and very good for the 3D distance between the two landmarks (r = 0.94, p < 0.001). CONCLUSION: 3D SPECT/CT image fusion is feasible, reproducible and allows correct superposition of SPECT segments onto cardiac CT anatomy.