Changes in brain extracellular glutamate during movement stress were studied using in vivo microdialysis. Male Long-Evans rats were placed in a clear cylinder designed to elicit behavioral activation while undergoing microdialysis sampling from either the hippocampus, striatum or sensorimotor cortex. Glutamate levels were determined by high performance liquid chromatography with fluorescence detection in the dialysates taken before, during, and after exposure to the cylinder. Animals were in a behaviorally quiescent state before exposure to the cylinder, but they demonstrated increases in rearing, locomotion, and turning while in the cylinder. Dialysate glutamate levels were significantly enhanced in the samples taken while the rat was in the cylinder compared with samples taken before or after exposure to the cylinder. In a second study, rats were implanted with bilateral probes in the forelimb sensorimotor cortex, and one forelimb was immobilized by means of a plaster of paris cast. Glutamate, aspartate, serine, and taurine levels were quantified in casted animals. In casted animals, dialysate glutamate levels were lower on the side contralateral to the immobilized limb during both quiescence and movement stress. Aspartate and taurine, but not serine levels increased during movement stress in both the side contralateral and the side ipsilateral to the immobilized limb. These results suggest that there is extracellular overflow of glutamate and other neuroactive amino acids during spontaneous movement, and chronic disuse can suppress extracellular glutamate levels.