This chapter describes methods for the solubilization of mammalian phospholipase D activity from the brain membranes and its assay using a fluorescent substrate, namely, l-palmitoyl-2-[6-N-(7-nitrobenzo-2-oxa-l,3-diazol-4-yl)amino]caproylphosphatidylcholine (C6-NBD-PC). Phospholipase D is a phosphodiesterase that attacks the distal phosphodiester bond in phospholipids. Mammalian phospholipase D activity was unequivocally observed in rat brain tissue. This brain membrane-bound PLD activity was further characterized and distinguished from a related phospholipid base-exchange activity by its acidic pH optimum, its independence of Ca2+, and its activation by free fatty acids in vitro. C6-NBD-PC, a fluorescent substrate was used for assaying solubilized PLD activity, and offered significant advantages over employing 3H-labeled PC. In addition to the convenience of a water-soluble short-chain substrate, the new assay was more sensitive and safer. This assay measured high PLD activity in solubilized membranes isolated from Swiss/3T3 fibroblasts. C 6 -NBD-PC could also be utilized as a PLD substrate in assays of membrane-bound (nonsolubilized) PLD, where PLD activity was likewise dependent on sodium oleate. Thus, C 6 -NBD-PC was a substrate for both the hydrolytic and the transphosphatidylation reactions catalyzed by PLD.