Olson in our laboratory developed a cell-free system for reconstitution of phospholipase D activity. Activity was calcium dependent and required protein factors in both the plasma membrane and cytosol. Activation stimuli included both GTPgS and PMA, implying the participation of both a GTP-binding/regulatory protein and protein kinase C. Because phosphatidic acid is metabolically unstable, phospholipase D activity is assayed by transphosphatidylation, a reaction in which the phosphatidate is preferentially transferred from phosphatidylcholine to a primary alcohol e.g. to form phosphatidylethanol. This metabolically stable product is easily detected by thin layer chromatography due to its unique migration.