Evidence that L-glutamate is a neurotransmitter of corticofugal fibers was sought by measuring changes in several biochemical markers of neurotransmitter function after pericruciate (sensorimotor) ablations in cats. Two weeks after cortical ablation, samples from various brain regions were analyzed for high affinity uptake of glutamate, gama- aminobutyric acid (GABA), glycine, alanine, and tyrosine. Amino acid levels and the activity of choline acetyltransferase (CAT) also were determined. High affinity glutamate uptake is decreased relative to the opposite side in areas of the nervous system which lost a predominantly unilateral corticofugal projection. These areas include the caudate nucleus, ventrolateral thalamic nucleus, red nucleus, basis pontis, and cervical and lumbar spinal cord. No significant changes were found in the uptake of other amino acids or in CAT in these regions. Changes in the levels of amino acids were significant only in ventrolateral thalamus where there was a 33% decrease in glutamate on the deafferented side. The data suggest that L-glutamate is a neurotransmitter of corticofugal fibers to many subcortical areas related to motor control.