Phospholipase D (PLD) is activated in mammalian cells in response to a wide variety of stimuli. A rapid assay for agonist-activated PLD activity in cell extracts was developed, utilizing a fluorescent derivative of phosphatidylcholine as substrate. Utilization of the substrate was assessed following thin-layer chromatography of the reaction mixture. Hydrolysis products generated by phospholipases D, C, and A2 could be visualized in the same reaction. Phorbol ester and vasopressin increased PLD activity in intact A7r5 vascular smooth muscle cells, as measured by an isotopic labeling method. Using the in vitro fluorescent assay, enhanced PLD activity was detected in membranes prepared from A7r5 cells that had been treated with phorbol ester or vasopressin. The agonist-activated activity was independent of phosphorylation occurring during the course of the assay. PLD activity was detected, in varying amounts, in membranes prepared from a variety of different mouse tissues. These results show that a fluorescent assay can be used to rapidly assess the activity of PLD and other phosphatidylcholine-utilizing phospholipases in cell and tissue extracts. The effects of agonists on PLD activity can be retained and quantitated in a broken cell preparation, permitting characterization of the agonist-activated form of the enzyme.