The endocannabinoid anandamide (N-arachidonoylethanolamine) and other bioactive long-chain N-acylethanolamines are thought to be formed from their corresponding N-acylphosphatidylethanolamines by a specific phospholipase D (NAPE-PLD) in the brain as well as other tissues. However, regional distribution of NAPE-PLD in the brain has not been examined. In the present study, we investigated the expression levels of NAPE-PLD in nine different regions of rat brain by enzyme assay, western blotting and real-time PCR. The NAPE-PLD activity was detected in all the tested brain regions with the highest activity in thalamus. Similar distribution patterns of NAPE-PLD were observed at protein and mRNA levels. We also found a remarkable increase in the expression levels of protein and mRNA of the brain NAPE-PLD with development, which was in good agreement with the increase in the activity. The age-dependent increase was also seen with several brain regions and other NAPE-PLD-enriched organs (heart and testis). p-Chloromercuribenzoic acid and cetyltrimethylammonium chloride, which inhibited recombinant NAPE-PLD dose-dependently, strongly inhibited the enzyme of all the brain regions. These results demonstrated wide distribution of NAPE-PLD in various brain regions and its age-dependent expression, suggesting the central role of this enzyme in the formation of anandamide and other N-acylethanolamines in the brain.