Dog brain homogenates and subcellular preparations incubated in the presence of Ca2+ produced a new phospholipid that was isolated and identified by its infrared spectrum and by chemical degradation as a mixture of 1,2-diacyl, alkenylacyl, and alkylacyl sn-glycero-3-phospho(N-acyl)ethanolamines, 50, 45, and 5%, respectively. The N-acyl groups consisted almost exclusively of 16:0, 18:0, and 18:1 fatty acids. Formation of N-acylethanolamine phospholipids from endogenous substrates was linear for about 90 min at approximately 4.5 nmol/h/mg protein and exhibited a pH optimum of 10. Biosynthetic activity was associated with particulate fractions, primarily microsomes, synaptosomes, and mitochondria, but not with myelin. In each case, small amounts (approximately 0.5 nmol/h/mg protein) of long-chain N-acylethanolamines were also produced. Incubation of dog brain microsomes with 1,2-di[1'-14C]palmitoyl glycerophosphocholine yielded N-acylethanolamine phospholipids labeled at both N-acyl (55%) and O-acyl (45%) moieties. It appears that dog brain organelles may contain a phosphatidylethanolamine N-acyl transferase (transacylase) analogous to that recently demonstrated in the myocardial tissue.