Single-photon emission tomography (SPET) using radioligands that are fixed on first pass through the cerebral circulation shows distinctive and rapidly changing blood flow patterns accompanying temporal lobe seizures. We sought to determine the optimal time to perform SPET studies for clinical seizure lateralization in temporal lobe epilepsy. Interictal, ictal and postictal SPET scans of 73 consecutively studied patients with unilateral temporal lobe epilepsy were read by three blinded observers to assess the accuracy of lateralization in each condition. The blinded observers correctly identified the side of focus in 97% of ictal studies, 72% of postictal studies and 50% of interictal studies. No incorrect scores were made in the ictal studies, whilst 5% of postictal and 12% of interictal studies were lateralized to the wrong side. Inter-observer agreement was best with the ictal studies. The dramatic perfusion changes of ictal SPET provide consistent, reliable and easily interpretable information that is superior to that provided by interictal and postictal studies. Injection of ligand during seizures is therefore the method of choice for SPET to aid the non-invasive lateralization of temporal seizure foci.