Growing rats were fed graded levels of threonine (Thr, 0.4, 0.8, and 3.3 g/100 g diet). Free amino acid content was measured in plasma and brain. Extracellular amino acid levels were measured by microdialysis in brain slices. Large quantities of dietary Thr (3.3 g/100 g) raised plasma and brain Thr and glycine (Gly) levels. Brain and spinal cord extracellular levels of Thr were also raised, whereas the other amino acid levels remained unchanged. A moderate level of dietary Thr (0.8 g/100 g) raised plasma Thr and Gly levels and brain Thr but not Gly level. The diet raised cortical Thr extracellular levels but did not modify the levels of the other amino acids, including glutamate (Glu) and aspartate (Asp). These data suggest that brain neurochemical processes involving Gly, Glu, and Asp are safeguarded in rats fed high Thr levels.