We have investigated whether the signaling protein phospholipase D is implicated in leukocyte cell motility. Treating differentiated HL-60 cells with small interfering RNAs (siRNAs), to deplete endogenous expression of the PLD1 isoform, led to an abolishment of basal chemokinesis that could not be rescued with chemoattractants ENA-78, FMLP, and IL-8. Transient overexpression of PLD1 increased both chemokinesis and chemotaxis toward IL-8 and FMLP but not toward ENA-78. Chemokinesis was not mediated by the enzymatic activity of PLD1, but the chemotactic response was, because a lipase-inactive mutant (PLD1-K830R) negated all chemokine-induced potentiating actions and because IL-8 and FMLP increased activity in vitro. Gene expression silencing of the other mammalian isoform, PLD2, also led to cell migration arrest, whereas ENA-78 selectively increased endogenous PLD2 activity and chemotaxis of HL-60 cells overexpressing a myc-pcDNA-PLD2 construct. Thus, PLD1 is differentially activated by CXCR-1, whereas CXCR-2 (and possibly CXCR-1) mediates PLD2 activation. Finally, immunofluorescence microscopy showed that both isoforms were associated with cell polarity and directionality concomitantly with adhesion and F-actin polymerization in response to IL-8. These data represent the first demonstration of the involvement of PLD and its enzymatic activity toward chemokines in the key physiologic process of leukocyte migration.