We performed localized 31P nuclear magnetic resonance (NMR) 1H-image-guided in vivo spectroscopy to study regional high-energy phosphate levels in the brains of normal controls and in patients with intractable unilateral temporal lobe epilepsy. We did not observe differences in intracellular pH between controls and patients. The phosphocreatine/inorganic phosphate ratio was reduced by 50% in the epileptogenic temporal lobe compared with controls (p < 0.005) and by 35% when compared with the unaffected contralateral temporal lobe (p < 0.05). We did not observe differences in the ratio of phosphomonoesters to phosphodiesters between controls and patients. These findings suggest that in vivo 31P NMR spectroscopy yields a distinctive interictal metabolic profile in patients with intractable unilateral temporal lobe epilepsy and may permit noninvasive lateralizing evidence of the seizure focus.