The ketogenic diet has multiple beneficial effects not only in treatment of epilepsy, but also in that of glucose transporter 1 deficiency, cancer, Parkinson's disease, obesity and pain. Thus, there is an increasing interest in understanding the mechanism behind this metabolic therapy. Patients on a ketogenic diet reach high plasma levels of ketone bodies, which are used by the brain as energy substrates. The interaction between glucose and ketone bodies is complex and there is still controversy as to what extent it affects the homeostasis of the neurotransmitters glutamate, aspartate and GABA. The present study was conducted to study this metabolic interaction in cultured GABAergic neurons exposed to different combinations of (13)C-labeled and unlabeled glucose and ß-hydroxybutyrate. Depolarization was induced and the incorporation of (13)C into glutamate, GABA and aspartate was analyzed. The presence of ß-hydroxybutyrate together with glucose did not affect the total GABA content but did, however, decrease the aspartate content to a lower value than when either glucose or ß-hydroxybutyrate was employed alone. When combinations of the two substrates were used (13)C-atoms from ß-hydroxybutyrate were found in all three amino acids to a greater extent than (13)C-atoms from glucose, but only the (13)C contribution from [1,6-(13)C]glucose increased upon depolarization. In conclusion, ß-hydroxybutyrate was preferred over glucose as substrate for amino acid synthesis but the total content of aspartate decreased when both substrates were present. Furthermore only the use of glucose increased upon depolarization.