Plasmalogens are ether-linked phospholipids highly abundant in nervous tissue. Previously we demonstrated that acute administration of myo-inositol (myo-Ins) + [2-(13)C] ethanolamine ([2-(13)C]Etn) significantly elevated phosphatidylethanolamine plasmalogen (PlsEtn) in rat whole brain. Current experiments investigated the effects of acute myo-Ins+[2-(13)C]Etn administration on [PlsEtn] and the biosynthesis of new Etn lipids using NMR spectroscopy in rat cerebral cortex, hippocampus, brainstem, midbrain and cerebellum. Treated rats received a single dose of myo-Ins + [2-(13)C]Etn and controls received saline rather than myoIns. Data reveal that the cerebellum is the brain region most affected by treatment, which resulted in a 22% increase in [PlsEtn] and 89% increase in newly synthesized Etn lipids relative to controls (P < 0.05). Furthermore, the cerebellar PlsEtn/phosphatidylethanolamine ratio and molar percentage of PlsEtn were significantly elevated by 12% and 8%, respectively (P < 0.05). These data suggest that myo-Ins influences Etn lipid metabolism in brain, particularly in the cerebellum where there is a stimulation in the biosynthesis of new Etn lipids with a preference towards PlsEtn.