Abstract Hereditary canine spinal muscular atrophy (HCSMA) is a lower motor neuron disease found in Brittany Spaniels that shares clinical and pathological features with human amyotrophic lateral sclerosis (ALS). Since acidic excitatory amino acids and the neuropeptide N-acetylaspartyl-glutamate (NAAG) are reduced in spinal cord and cerebral cortex in ALS, the levels of these substances were measured in nervous tissue in Brittany Spaniels heterozygous and homozygous for HCSMA. Significant reductions in the levels of endogenous aspartate, glutamate, N-acetylaspartate (NAA), and NAAG were found in the spinal cord in homozygous but not heterozygous HCSMA. In contrast, the activity of N-acetylated-α-linked-amino dipeptidase (NAALADase), an enzyme that cleaves NAAG into NAA and Glu, was significantly increased. None of these parameters was affected in the motor cortex or occipital cortex. Since NAA and NAAG are highly concentrated in motoneurons, they may play a role in the pathogenesis of motor neuron disease.