There is now increasing evidence that phospholipase D (PLD) activity can be stimulated by a range of hormones, growth factors, and neurotransmitters in a range of cell types (1 ). The enzyme generally catalyzes the hydrolysis of phosphatidylcholine (PtdCho) to produce phosphatidate (PtdOH) and choline (Cho), although the hydrolysis of phosphatidylethanolamine has been reported. PtdOH can be converted to diacylglycerol (DAG) by the action of phosphatidate phosphohydrolase, and the PLD pathway has thus been suggested to provide a source of DAG in cells. This reaction clearly takes place, however, whether it provides a DAG, which has a signaling function, remains unclear. This chapter describes methods to determine PLD activity and also to examine the levels of both PtdOH and DAG.