Although oleate has been implicated in the regulation of phospholipase D (PLD) activity, the molecular identity of the oleate-stimulated PLD is still poorly understood. We now report that oleate selectively stimulates the enzymatic activity of PLD2 but not of PLD1, with an optimal concentration of 20 microM in vitro. Intriguingly, phosphatidylinositol 4,5-bisphosphate (PIP2) synergistically stimulates the oleate-dependent PLD2 activity with an optimal concentration of 2.5 microM. These results provide the first evidence that oleate is a PLD2-specific activating factor and PLD2 activity is synergistically stimulated by oleate and PIP2.