Study design: The effect of long-term (4 weeks) moderate locomotor exercise on segmental distribution of glutamate (Glu), aspartate, gamma-aminobutyric acid, glycine (Gly), serotonin and noradrenaline in the spinal cord of adult rats was investigated. Objectives: In light of the data showing modulation of some neurotransmitters in the lowlumbar segments of the rat due to physical exercise, our aim was to establish how segmentally specific is this effect with respect to neuroactive amino acids and monoamines. Setting: Laboratory of Reinnervation Processes, Department of Neurophysiology, Nencki Institute of Experimental Biology, Warsaw, Poland. Methods: Amino acids and monoamines content was measured by means of HPLC in the whole tissue homogenate of the spinal cord in nonexercised and exercised rats. Results: Glu and Gly homogenate concentration was the highest among all tested compounds. There was an intersegmental rostro-caudal gradient of concentration of neuroactive amino acids and monoamines, progressing caudally. Exercise modified this gradient exerting opposite effect on their concentration of amino acids and monoamines in the rostral and caudal lumbar segments. Conclusion: Locomotor exercise leads to neurochemical remodeling of the spinal cord, which is differently manifested in the rostral and caudal lumbar segments of the spinal cord.