Abstract Purpose: To compare the sensitivities of MRI, FDG-PET and ictal/SPECT in localization of the epileptogenic substrate in patients with refractory temporal lobe epilepsy. Patients and methods: This study included 137 patients who received surgical treatment for intractable epilepsy. MRI, FDGPET and ictal 99mTc-HMPAO SPECT were retrospectively reviewed regarding their sensitivity in lateralization of the epileptogenic zone compared to video/EEG, pathological results and surgical outcome. Results: 104 MR-positive and 33 MR-negative patients were enrolled. In the MR-positive group, MRI, PET and ictal/SPECT were concordant to video/EEG in 72%, 83% and 73%, respectively. When compared to pathological diagnosis, they correctly lateralized the epileptogenic zone in 70%, 87%, and 73%, respectively. In patients with good surgical outcome, they correctly localized the epileptogenic zone in 79%, 88%, and 78%, respectively. In the MR-negative group, PET andictal/SPECT were concordant with video/EEG in 82% and 58%, respectively and matching with pathological diagnosis in 85% and 56%, respectively. Conclusion: PET is the most sensitive method in lateralization of the epileptogenic substrate. The use of MRI, PET and ictal/SPECT as a multimodality approach improves lateralization of the affected zone particularly in cases with negative MR findings and distinguishes patients who will benefit from surgery.