N-Acyl-phosphatidylethanolamines (NAPEs), a minor class of membrane glycerophospholipids, accumulate along with their bioactive metabolites, N-acylethanolamines (NAEs) during ischemia. NAPEs can be formed through N-acylation of phosphatidylethanolamine by cytosolic phospholipase A₂ε (cPLA₂ε, also known as PLA2G4E) or members of the phospholipase A and acyltransferase (PLAAT) family. However, the enzyme responsible for the NAPE production in brain ischemia has not yet been clarified. Here, we investigated a possible role of cPLA₂ε using cPLA₂ε-deficient (Pla2g4e^−/−) mice. As analyzed with brain homogenates of wild-type mice, the age dependency of Ca²⁺-dependent NAPE-forming activity showed a bell-shape pattern being the highest at the first week of postnatal life, and the activity was completely abolished in Pla2g4e^−/− mice. However, liquid chromatography-tandem mass spectrometry revealed that the NAPE levels of normal brain were similar between wild-type and Pla2g4e^−/− mice. In contrast, post-mortal accumulations of NAPEs and most species of NAEs were only observed in decapitated brains of wild-type mice. These results suggested that cPLA₂ε is responsible for Ca²⁺-dependent formation of NAPEs in the brain as well as the accumulation of NAPEs and NAEs during ischemia, while other enzyme(s) appeared to be involved in the maintenance of basal NAPE levels.