A robust algorithm to disclose and display the difference of ictal and interictal perfusion may facilitate the detection of ictal hyperfusion foci. Diagnostic performance of localizing epileptogenic zones with subtracted SPECT images was compared with the visual diagnosis using ictal and interictal SPECT, MR, or PET. Ictal and interictal Tc-99m-HMPAO cerebral perfusion SPECT images of 48 patients(pts) were processed to get parametric subtracted images. Epileptogenic foci of all pts were diagnosed by seizure free state after resection of epileptogenic zones. In subtraction SPECT, we used normalized difference ratio of pixel counts((ictal-interictal)/interictal¡¿100%) after correcting coordinates of ictal and interictal SPECT in semi-automatized 3-dimensional fashion. We found epileptogenic zones in subtraction SPECT and compared the performance with visual diagnosis of ictal and interictal SPECT, MR and PET using post-surgical diagnosis as gold standard. The concordance of subtraction SPECT and ictal-interictal SPECT was moderately good(kappa=0.49). The sensitivity of ictal- interictal SPECT was 73% and that of subtraction SPECT 58%. Positive predictive value of ictal-interictal SPECT was 76% and that of subtraction SPECT was 64%. There was no statistical difference between sensitivity or positive predictive values of subtraction SPECT and ictal-interictal SPECT, MR or PET. Such was also the case when we divided patients into temporal lobe epilepsy and neocortical epilepsy. We conclude that subtraction SPECT we produced had equivalent diagnostic performance compared with ictal-interictal SPECT in localizing epileptogenic zones. Additional value of these subtraction SPECT in clinical interpretation of ictal and interictal SPECT should be further evaluated.