Phosphatidic acid (PA) is a critical phospholipid constituent in eukaryotic cell membranes, which accounts for 1À4% of the total lipid. 1 This lipophilic glycerophospholipid has a phosphate headgroup, and as such serves not only a structural capacity in lipid bilayers, but also participates as an intermediate in lipid metabolism and as a signaling molecule. Because of the small headgroup, PA facilitates changes in lipid bilayer curvature that are important for membrane fusion events, such as vesicular trafficking and endocytosis. 2 PA is also a precursor to other lipid signaling molecules including diacylglycerol (DAG) and lysophosphatidic acid (LPA). As a lipid second messenger, PA activates signaling proteins and acts as a node within the membrane to which signaling proteins translocate. Several signaling proteins, including Raf-13, 4 and mTOR, 5 directly bind PA to mediate translocation or activation, respectively. PA has been implicated in signaling cascades involving cell growth, proliferation, and survival. Aberrant PA signaling has been identified in multiple cancers, 6 neurodegeneration, 7 and platelet aggregation, 8 which makes proteins that mediate cellular levels of PA attractive as potential therapeutic targets. PA can be generated de novo9À11 by sequential enzymecatalyzed acylations of glycerol-3-phosphate, or in response to cell signaling pathways (Figure 1). Every glycerophospholipid generated in eukaryotic membranes transitions through PA, a pathway characterized by Eugene Kennedy and his colleagues more than half a century ago. 11, 12 Signal generated PA is formed by enzymes that modify existing lipids. These enzymes include lysophosphatidic acid acyltransferase (LPAAT), which acylates LPA, DAG kinase, which phosphorylates DAG at the sn-3 position, and phospholipase D (PLD), which hydrolyzes the headgroup of a phospholipid, generally phosphatidylcholine (PC), triggering the release of choline. PLD activity, an enzyme-catalyzed hydrolysis of a phosphodiester bond, was first described in plants, 13À16 and subsequently many enzymes from a range of viral, prokaryotic, and eukaryotic organisms have been described as possessing PLD activity. To date, more than 4000 PLD enzymes have been entered in NCBI GenBank. The majority of these enzymes hydrolyze phosphodiester bonds within phospholipids such as PC (classified as EC 3.1. 4.417), but there are other enzymes ascribed to having PLD activity that hydrolyze neutral lipids and even polynucleotide backbone. A large subset of enzymes with PLD activity share a conserved HxKxxxxDx6GSxN motif (HKD), 18 or a variation thereof, which is responsible for catalytic activity. These enzymes are members of the PLD superfamily and are proposed to follow a similar reaction mechanism. Non-HKD enzymes exhibiting PLD activity have divergent structures and catalytic mechanisms. These non-HKD enzymes are discussed here as a means of comparison. In this comprehensive Review of the PLD superfamily, specific emphasis is given to the conventional mammalian isoforms, PLD1 and PLD2, and the tools with which these enzymes are studied. The merits of PLD as a potential therapeutic target are also reviewed, as are implications for modulation of PLD activity in cell signaling pathways, whole organisms, and aberrant or disease-related models.