Phospholipase D (PLD) is an enzyme that hydrolyzes phosphatidylcholine yielding choline and phosphatidic acid. PLD is activated by mitogens (lead to cell division) and motogens (leading to cell migration). PLD is known to contribute to cellular proliferation and deregulated expression of PLD has been implicated in several human cancers. PLD has been found to play a role in leukocyte chemotaxis and adhesion as studied through the formation of chemokine gradients. We have established a model of cell migration comprising three cell lines: macrophages RAW 264.7 and LR-5 (for innate defense), and fibroblast COS-7 cells (for wound healing). COS-7 cells respond to EGF, while the other cell lines respond to MIP-1a and MCP-1. Transfection of cells with either PLD1-WT or PLD2-WT constructs leads to increased cell chemotaxis. PLD2-WT is better (=1.5-fold) at increasing chemotaxis than PLD1 WT. Phospholipase inactive mutants in the HKD domain have a negative effect on chemotaxis. Two PLD2 mutants in the PX domain, Y169F and Y179F, known to inhibit the ability of PLD2 to bind through an SH2 domain, failed to potentiate chemotaxis. Conversely, mutation created near, but not in, the SH2 recognition domain in PLD2 (PLD2 Y165F) did not impair the positive effect on chemotaxis observed for the WT. We have demonstrated also here that a proteinprotein interaction between PLD2 and Grb2 is needed to enhance chemotaxis. Thus, PLD, signaling through Grb2, is a key regulator of the functionality of the three cells studied. This may pay an important role in facilitating wound healing and innate defense capabilities of our body.