The 16 kD subunit of vacuolar H+-ATPase (V-ATPase) is essential for pumping protons into intracellular organella. Northern blot analysis revealed that the 16 kD subunit of V-ATPase is abundantly expressed in rat brain, and that the amount of transcripts is higher in the postnatal 15 days than in the adult age. On in situ hybridization, the mRNA signal for 16 kD subunit of V-ATPase was detected widely over the brain tissues of both adult and postnatal rats, with higher intensity in most of the gray matters located in the cerebellar cortex, hippocampus, thalamic nuclei, pontin nuclei, and olfactory bulb. In contrast, the signal in the cerebral cortex was preferentially enriched in the surface layers in the postnatal 10 days but was distributed diffusely and weakly over the entire layers thereafter. In all regions of brain, the mRNA signal was located predominantly in the neurons rather than in the glial components.