Background: Temporal lobe epilepsy (TLE) is the most frequent cause of focal and refractory seizures. Magnetic Resonance Spectroscopy (MRS) is a potentially useful tool in the investigation of brain metabolites in TLE. Objective: to clarify the role of MRS in the TLE epilepsy and to evaluate the relationship between the results of MRS, Electroencephalography (EEG), and the clinical data of TLE patients with negative Magnetic Resonance imaging (MRI). Patients & Methods: Thirty patients with TLE and negative MRI were investigated by EEG, MRS and neuropsychological tests that assessed memory function of temporal lobe. By using temporal metabolite identified on MRS, we could detect metabolic disturbance that occur in TLE and tried to lateralize epileptic focus. We correlated MRS results with, EEG results, and the patient's scores on memory function tests. Twenty age-matched volunteers were used as control subjects. Result(s): Ipsilateral temporal N-acetyl-aspartate (NAA)/ Creatine+Choline (Cr+Cho) and NAA/Cr ratios of the patients were significantly reduced compared with the mean ratios of contralateral hippocampus (p <0.001) and control subjects (p <0.001). EEG could detect epileptic focus in 17 (56.7%) patients, while MRS could detect epileptic focus in 27 (90%) patients and could lateralize about 76.9% of patients with bitemporal interictal epileptiform discharges (IEDs). Significant negative correlation between ipsilateral temporal NAA/Cr+Cho, NAA/Cr ratios and ipsilateral IEDs ratio (p < 0.05). Left temporal NAA/Cr ratio correlated significantly with patient's scores on Rey Auditory Verbal Learning Test (RAVLT) (p< 0.05) and right temporal NAA/Cr ratio correlated significantly with patient's scores on Rey Complex Figure Test (RCFT) (p < 0.05). Conclusion(s): These findings suggested that the temporal metabolite ratios identified on MRS could lateralize epileptic focus in TLE patients and they are closely related to the temporal IEDs and the cognitive function of temporal lobe.