N-Acyl-phosphatidylethanolamine (NAPE) is present in very small amounts in mammalian tissues (less than 0.1% of total phospholipids). However, NAPE as well as its degradation product, N-acylethanolamine (NAE), can be formed in certain neuronal tissues in response to increased [Ca2+]i. A high [Ca2+]i will activate the NAPE-forming N-acyltransferase using the sn-1 acyl group of a donor phospholipid as substrate in the transfer reaction. This membrane-bound enzyme seems to have no substrate specificity with respect to transfer of acyl groups; thus the fatty acids in the N-acyl group of NAPE are mainly 16:0 and 18:1, corresponding to the fatty acids in the sn-1 acyl group of the donor phospholipids. The NAPE-hydrolyzing phospholipase D also seems not to be acyl-group specific. In mouse neocortical neurons in primary culture, formation of NAPE and NAE is stimulated by glutamate via activation of the N-methyl-D-aspartate-receptor. Both NAPE and, to a lesser extent, NAE accumulate in a linear fashion for many hours while at the same time the neurons are dying. Likewise, in neurons prelabeled with 14C-arachidonic acid, 14C-arachidonic acid-labeled NAPE, and anandamide (= N-arachidonoylethanolamine) are accumulating. The formation of NAPE and NAE may represent a cytoprotective response in relation to various forms of neurotoxicity.