Phospholipase D (PLD) hydrolyzes phospholipids into phosphatidic acid (PA) and the base head groups. In mammalian cells, this activity plays an active role in signal transduction by acting as a mediator for a variety of extracellular stimuli. Two mammalian PLD genes have been identified and the encoded enzymes expressed and characterized. The activity of PLD1 is regulated by the ADPribosylation factor (ARF) and Rho families of monomeric GTPases, classic isoforms of protein kinase C (PKC), and the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP 2 ). PLD2, although highly sensitive to PIP 2 has shown only modest response to ARF and appears unresponsive to Rho and PKC, in vitro . Characterization of PLD activity and identification of various regulatory molecules were greatly facilitated by the establishment of an assay for in vitro studies. Alternative methods for the assay of PLD described in this chapter facilitate measurement of basal activity and regulation in the absence of PIP 2 .