In animal tissues anandamide and other bioactive N-acylethanolamines are principally produced from glycerophospholipids through the transacylation-phosphodiesterase pathway consisting of two enzymatic reactions. The first reaction is the generation of N-acylphosphatidylethanolamine (NAPE) by transferring an acyl group esterified at sn-1 position of glycerophospholipid to the amino group of phosphatidylethanolamine. This reaction is catalyzed by Ca(2+)-dependent N-acyltransferase. The discovery of Ca(2+)-independent N-acyltransferase revealed the existence of plural enzymes which are capable of catalyzing this reaction. The second reaction is the release of N-acylethanolamine from NAPE catalyzed by NAPE-hydrolyzing phospholipase D (NAPE-PLD). The enzyme belongs to the metallo-beta-lactamase family and specifically hydrolyzes NAPEs. Recent studies, including analysis of NAPE-PLD-deficient mice, led to the discovery of NAPE-PLD-independent pathways for the anandamide biosynthesis.