Phospholipase D (PLD) is distributed widely in nature, being present in various isoforms in bacteria, protozoa, fungi, plants, and animals. It catalyzes the hydrolysis of phospholipids, primarily phosphatidylcholine (PC), into phosphatidic acid (PA) and the head group, choline. It also catalyzes a transphosphatidylation reaction in which water is replaced by a primary alcohol to yield a phosphatidyl alcohol. This reaction is exclusive to PLD and is employed as a specific assay for the enzyme in in vivo systems. When the purified enzyme is assayed in vitro, the release of choline from PC can be utilized. This chapter describes production of a recombinant mammalian isozyme of PLD (PLD1) in baculovirus-infected insect cells and its purification. It also provides details of the assay procedure in the presence and absence of regulatory proteins in vitro. The assay of the enzyme in cells in vivo is also documented using labeling of endogenous PC by incubating the cells with (3)H-labeled fatty acid. Details of the assay utilizing the transphosphatidylation reaction are presented. In this, 1-butanol is employed as the primary alcohol and [(3)H]phosphatidylbutanol is isolated by thin-layer chromatography of lipid extracts from the cells. A variation of this assay is described using deuterated 1-butanol (1-butanol-d(10)) and detection of the synthesized deuterated phosphatidylbutanol species by mass spectrometry. Convenient alternative assays for PLD and diacylglycerol (DAG) lipase activity based on fluorescence are also described. Many of the materials for these assays are available commercially, with the exception of the fluorescently labeled DAG substrate, which can be synthesized enzymatically in a simple one-step procedure.