Peri-ictal single-photon emission tomography (SPET) difference images co-registered to magnetic resonance imaging (MRI) visualize regional cerebral blood flow (rCBF) changes and help localize the epileptogenic area in medically refractory epilepsy. Few reports have examined the reproducibility of SPET difference image results. Epilepsy patients having two peri-ictal and at least one interictal SPET scan who later underwent surgical resection were studied. Localization accuracy of peri-ictal SPET difference images results, interictal electroencephalography (EEG), and ictal EEG from the first (seizure 1) and second (seizure 2) seizure, as well as MRI and positron emission tomography (PET) findings, were compared using surgical resection site as the standard. Thirteen patients underwent surgical resection (11 temporal lobe and 2 extratemporal). SPET results from seizure 1 were localized to the surgical site in 12/13 (92%) patients, while SPET results from seizure 2 were localized in 13/13 (100%) patients. All other modalities were less accurate than the SPET results interictal EEG--seizure 1 6/13 (46%); ictal EEG--seizure 1 5/13 (38%); interictal intracranial EEG--seizure 2 4/9 (44%); ictal intracranial EEG--seizure 2 results 8/9 (89%); MRI 6/13 (46%); PET 9/13 (69%)[. SPET results were reproducible in 12/13 (92%) patients. SPET difference images calculated from two independent peri-ictal scans appear to be reproducible and accurately localize the epileptogenic area. While SPET difference images visualize many areas of rCBF change, the quantification of these results along with consideration of injection time improves the diagnostic interpretation of the results.