The effects of acetyl-L-carnitine on cerebral glucose metabolism were investigated in rats injected with differently 14C- and 13C-labelled glucose and sacrificed after 15, 30, 45, and 60 min. Acetyl-L-carnitine was found to reduce total 14CO2 release from [U-14C]glucose along with the decrease in [1-13C]glucose incorporation into cerebral amino acids and tricarboxylic acid cycle intermediates. However the 13C labelling pattern within different carbon positions of glutamate, glutamine, GABA, and aspartate was unaffected by acetyl-L-carnitine administration. Furthermore, the cerebral levels of newly-synthesized proglycogen were higher in rats treated with acetyl-L-carnitine than in untreated ones. These results suggest that acetyl-L-carnitine was able to modulate cerebral glucose utilization and provide new insights on the mechanisms of action of this molecule in the central nervous system.