Oleate-dependent phospholipase D (PLD; EC 3.1.4.4) has been reported in animal systems, but its molecular nature is unkown. Multiple PLDs have been characterized in plants, but none of the previously cloned PLDs exhibits the oleate-activated activity. Here, we describe the biochemical and molecular identification and characterization of an oleate-activated PLD in Arabidopsis. This PLD, designated PLDdelta, was associated tightly with the plasma membrane, and its level of expression was higher in old leaves, stems, flowers, and roots than in young leaves and siliques. A cDNA encoding the oleate-activated PLD was identified, and catalytically active PLDdelta was expressed from its cDNA in Escherichia coli. PLDdelta was activated by free oleic acid in a dose-dependent manner, with the optimal concentration being 0.5 mM. Other unsaturated fatty acids, linoleic and linolenic acids, were less effective than oleic acid, whereas the saturated fatty acids, stearic and palmitic acids, were totally ineffective. Phosphatidylinositol 4,5-bisphosphate stimulated PLDdelta to a lesser extent than oleate. Mutation at arginine (Arg)-611 led to a differential loss of the phosphatidylinositol 4,5-bisphosphate-stimulated activity of PLDdelta, indicating that separate sites mediate the oleate regulation of PLDdelta. Oleate stimulated PLDdelta's binding to phosphatidylcholine. Mutation at Arg-399 resulted in a decrease in oleate binding by PLDdelta and a loss of PLDdelta activity. However, this mutation bound similar levels of phosphatidylcholine as wild type, suggesting that Arg-399 is not required for PC binding. These results provide the molecular information on oleate-activated PLD and also suggest a mechanism for the oleate stimulation of this enzyme.