The N-acylphosphatidylethanolamine-hydrolysing phospholipase D (NAPE-PLD) generates N-acylethanolamines, including N-arachidonoyl-ethanolamine (anandamide), that may be neuroprotective and analgesic. The properties of NAPE-PLD from rat heart and brain microsomes are investigated and compared to those of other PLDs. NAPE-PLD is inhibited by the fatty acid aminohydrolase inhibitor MAFP in high concentrations (=100 µM) while PMSF in high concentrations (10 mM) tends to stabilise NAPE-PLD activity. Oleate inhibits NAPE-PLD but the enzyme is not affected by PIP, a-synuclein or mastoparan. Furthermore, it is for the first time reported that NAPE-PLD is not capable of catalysing a transphosphatidylation reaction like most other known PLDs. Copyright (C) 1999 Federation of European Biochemical Societies.