Glycerol is effective in the treatment of brain oedema but it is unclear if this is due solely to osmotic effects of glycerol or whether the brain may metabolize glycerol. We found that intracerebral injection of [14C]glycerol in rat gave a higher specific activity of glutamate than of glutamine, indicating neuronal metabolism of glycerol. Interestingly, the specific activity of GABA became higher than that of glutamate. NMR spectroscopy of brains of mice given 150 micromol [U-13C]glycerol (0.5 m i.v.) confirmed this predominant labelling of GABA, indicating avid glycerol metabolism in GABAergic neurones. Uptake of [14C]glycerol into cultured cerebellar granule cells was inhibited by Hg2+, suggesting uptake through aquaporins, whereas Hg2+ stimulated glycerol uptake into cultured astrocytes. The neuronal metabolism of glycerol, which was confirmed in experiments with purified synaptosomes and cultured cerebellar granule cells, suggested neuronal expression of glycerol kinase and some isoform of glycerol-3-phosphate dehydrogenase. Histochemically, we demonstrated mitochondrial glycerol-3-phosphate dehydrogenase in neurones, whereas cytosolic glycerol-3-phosphate dehydrogenase was three to four times more active in white matter than in grey matter, reflecting its selective expression in oligodendroglia. The localization of mitochondrial and cytosolic glycerol-3-phosphate dehydrogenases in different cell types implies that the glycerol-3-phosphate shuttle is of little importance in the brain.