We performed proton magnetic resonance spectroscopic imaging (1H-MRSI) in patients with motor neuron disease (MND) to evaluate the distribution and extent of cortical neuron damage or loss as reflected by decreased N-acetyl (NA) to creatine (Cr) resonance intensity ratios. We examined premotor (superior frontal gyrus), primary motor (precentral gyrus), primary sensory (postcentral gyrus), and parietal (superior parietal gyrus/precuneus) neocortical regions of 12 patients with MND and six normal control subjects. Patients with MND were representative of three syndromes: amyotrophic lateral sclerosis (ALS) with definite lower motor neuron and upper motor neuron signs, MND with probable upper motor neuron signs (PUMNS), and progressive spinal muscular atrophy (PSMA) with lower motor neuron signs only. Compared with healthy controls, ALS patients had a significant decrease in NA/Cr resonance intensity ratios, most prominently in the primary motor cortex (p < 0.001) but also, to varying degrees, in primary sensory (p < 0.01), posterior premotor, and parietal (p < 0.05) regions. Patients classified as ALS-PUMNS showed less prominent reduction in NA/Cr ratios in the same regions; patients with PSMA had normal cortical NA/Cr ratios. Sequential studies in one patient suggested that 1H-MRSI could document progression of the NA/Cr abnormality. Decreased NA/Cr ratios on 1H-MRSI provide an index of cortical motor neuron loss and/or dysfunction in MND patients. Clinical applications of 1H-MRSI could include documenting the extent of upper motor neuron involvement, aiding diagnosis of syndromes presenting with an ALS-like picture, and monitoring disease progression.