We have evaluated a three-dimensional localized spectroscopic imaging sequence that uses two pairs of adiabatic full-passage pulses, which optimizes the detection of glutamate resonances at moderate echo times. This sequence provides excellent volume localization while simultaneously reducing J-modulation losses of glutamate. We have simulated the performance of this sequence for glutamate and used it to quantitatively measure glutamate in the human hippocampus using a linear components model. Using tissue segmentation and regression analysis, we measured a glutamate concentration of 8.8 ± 2.1 mM in hippocampal and temporal gray matter and 3.7 ± 1.1 mM in temporal white matter (95% CI). We have used this approach in a small group of patients (n = 5) with unilateral hippocampal epilepsy. Copyright © 2006 John Wiley & Sons, Ltd.