Publisher Summary This chapter discusses the detection and characterization of the soluble phospholipase D. Phospholipase D is an important component in cellular signal transduction mechanisms. A unique property of phospholipase D is its ability to catalyze a transphosphatidylation reaction by which the phosphatidyl moiety of the phospholipid substrate is transferred to a nucleophilic acceptor. The enzyme is activated in many cells in response to a wide variety of receptor agonists. Phospholipase D catalyzes the hydrolysis of phospholipid substrates to generate phosphatidic acid and water-soluble bases. Phosphatidic acid and its dephosphorylated product diacylglycerol are important intracellular messengers. For the characterization of phospholipase D, the phosphatidic acid and phosphatidylethanol are localized by iodine staining, and the area containing phosphatidic acid and phosphatidylethanol are scraped into scintillation vials. Scintillation fluid is added to scintillation vials, and silica gel particles are suspended. The radioactivity in the vials is quantified by liquid scintillation spectrometry. Using phosphatidylcholine as a substrate, the effect of fatty acid composition in the activity is evaluated. The nature of the fatty acyl moieties in positions 1 and 2 exerted a profound effect on phospholipase D activity. Phosphatidylcholine with arachidonic acid at position 2 and palmitic acid at position 1 is the most preferred substrate.