Phospholipase D (PLD), which catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid and choline, plays key roles in cellular signal transduction by mediating extracellular stimuli including hormones, growth factors, neurotransmitters, cytokines and extracellular matrix molecules. The molecular mechanisms by which domains regulate the activity of PLD--especially the phox homology (PX) domain--have not been fully elucidated. In this study, we have examined the properties of the PX domains of PLD1 and PLD2 in terms of phosphoinositide binding and PLD activity regulation. Interestingly, the PX domain of PLD1, but not that of PLD2, was found to specifically interact with phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3). We found that mutation of the conserved arginine at position 179 of the PLD1 PX domain to lysine or to alanine (R179A or R179K, respectively) disrupts PtdIns(3,4,5)P3 binding. In NIH-3T3 cells, the EGFP-PLD1 PX wild-type domain, but not the two mutants, localized to the plasma membrane after 5-minute treatment with platelet-derived growth factor (PDGF). The enzymatic activity of PLD1 was stimulated by adding PtdIns(3,4,5)P3 in vitro. Treatment with PDGF resulted in the significant increase of PLD1 activity and phosphorylation of the downstream extracellular signal-regulated kinases (ERKs), which was blocked by pre-treatment of HEK 293 cells with phosphoinositide 3-kinase (PI3K) inhibitor after the endogenous PLD2 had been depleted by siRNA specific for PLD2. Nevertheless, both PLD1 mutants (which cannot interact with PtdIns(3,4,5)P3) did not respond to treatment with PDGF. Moreover, PLD1 was activated in HepG2 cells stably expressing the Y40/51 mutant of PDGF receptor that is required for the binding with PI3K. Our results suggest that the PLD1 PX domain enables PLD1 to mediate signal transduction via ERK1/2 by providing a direct binding site for PtdIns(3,4,5)P3 and by activating PLD1.