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Objective: To outline and validate a technique for subtraction ictal single photon emission tomography (SPET) co-registered to magnetic resonance imaging (MRI) (SISCOM), which allows non-linear co-registration of groups of subtraction SPET images to a single, template image., Methods: In patients with mesial temporal lobe epilepsy, we used linear and non-linear transformation steps to co-register ictal and interictal images to a template SPET, then used the resultant ictal and interictal images to produce subtraction images. Statistical changes in subtraction SPET before and after the transformation steps in individual subjects were documented to validate the technique. Subtraction SPET images were thresholded to include pixel values 1 standard deviation (SD) above zero, and converted to binary. Images were combined by simple addition of images to form the final composite image. Final results were co-registered to a template MRI to demonstrate the regions most commonly hyperperfused in mesial temporal lobe epilepsy., Results: Linear and non-linear transformation steps induced only small changes in standard deviations of subtraction SPET, typically approximately 1 pixel value. The final composite SISCOM images showed the anterior temporal lobe, insula and basal ganglia as the most commonly hyperperfused regions during mesial temporal lobe epileptic seizures., Conclusion: Our technique resulted in only small changes in statistical characteristics of individual subtraction SPET studies, and was acceptable for the purpose of creating composite SISCOM images.