The relationship between ketosis and brain amino acid metabolism was studied in mice that consumed a ketogenic diet (>90% of calories as lipid). After 3 days on the diet the blood concentration of 3-OH-butyrate was ∼5 mmol/l (control = 0.06–0.1 mmol/l). In forebrain and cerebellum the concentration of 3-OH-butyrate was ∼10-fold higher than control. Brain [citrate] and [lactate] were greater in the ketotic animals. The concentration of whole brain free coenzyme A was lower in ketotic mice. Brain [aspartate] was reduced in forebrain and cerebellum, but [glutamate] and [glutamine] were unchanged. When [15N]leucine was administered to follow N metabolism, this labeled amino acid accumulated to a greater extent in the blood and brain of ketotic mice. Total brain aspartate (14N + 15N) was reduced in the ketotic group. The [15N]aspartate/[15N]glutamate ratio was lower in ketotic animals, consistent with a shift in the equilibrium of the aspartate aminotransferase reaction away from aspartate. Label in [15N]GABA and total [15N]GABA was increased in ketotic animals. When the ketotic animals were injected with glucose, there was a partial blunting of ketoacidemia within 40 min as well as an increase of brain [aspartate], which was similar to control. When [U-13C6]glucose was injected, the 13C label appeared rapidly in brain lactate and in amino acids. Label in brain [U-13C3]lactate was greater in the ketotic group. The ratio of brain 13C-amino acid/13C-lactate, which reflects the fraction of amino acid carbon that is derived from glucose, was much lower in ketosis, indicating that another carbon source, i.e., ketone bodies, were precursor to aspartate, glutamate, glutamine and GABA. J. Neurosci. Res. 66:272–281, 2001. © 2001 Wiley-Liss, Inc.