Single-voxel proton magnetic resonance spectroscopy (1H MRS) has shown abnormalities in patients with temporal lobe epilepsy (TLE) and hippocampal sclerosis (HS). Many TLE patients, however, do not have HS or other lesions on quantitative magnetic resonance imaging (MRI) (MRI-negative). Fifteen control subjects, 15 patients with unilateral HS, and 15 MRI-negative TLE patients underwent 1H MRS at an echo time of 30 msec on a 1.5-T GE Signa scanner. Voxels were tailored to the individual hippocampi. N-Acetylaspartate (NAA), creatine, choline, total glutamate plus glutamine (Glx), and myo-inositol (Ins) were quantitated by using an external standard and LCModel, a user-independent quantitation method. Normal ranges were defined as the control mean +/- 2.5 SD. In HS patients, 12 of 15 had abnormally low NAA in sclerotic hippocampi; 3 of these 12 also had abnormally low NAA contralaterally. Abnormally low NAA/Ins ratios lateralized the side affected by HS in 7 of 15 patients, without any bilateral abnormalities. In 15 MRI-negative TLE patients, 4 had abnormally low hippocampal NAA ipsilateral to seizure onset, 1 of whom had abnormally low NAA bilaterally. Analysis of groups of subjects showed a bilateral decrease in NAA, most marked in patients with HS and on the side of seizure onset. The mean NAA/Ins ratio was lower in patients with HS than in control subjects and in MRI-negative patients. The concentration of Glx was higher ipsilateral to seizure onset in MRI-negative patients than in HS patients. Quantitative short echo time 1H MRS identified abnormalities in 87% of patients with HS and 27% of MRI-negative TLE patients in concordance with other lateralizing data. In individual and group comparisons, 1H MRS described a metabolite profile in the hippocampi of MRI-negative TLE patients that was different from patients with HS, with an increase in Glx and a less marked decrease in NAA than was seen in HS.