Phospholipase D (PLD) plays a role in tumorgenesis in several cell lines through the formation of phosphatidic acid (PA) and its downstream metabolites. PLD is involved in proliferation and cytoskeleton rearrangements. Primary alcohol treatment blocks PA formation and is used to define PLD involvement in these events. Two mammalian PLD isoforms are known, PLD1 and PLD2. Both isoforms are regulated by small G proteins, protein interactions and protein kinases. At this time, PLD2 is thought to the predominant isoform involved in signaling. Our focus was to determine the PLD isoform responsible for mitogenic events. Phenylephrine (PE), an α1-adrenergic receptor agonist, leads to the activation of the ERK pathway, stress fiber formation and cell migration in Chinese Hamster lung fibroblasts (CCL39). Using dominant-negative (DN) PLD1 and PLD2, we determined that PLD1 is responsible for these actions. In earlier studies 50 μM PE stimulated ERK activity 3-5 fold in a PLD dependent fashion. Expression of DN PLD 1 but not DN PLD2 decreased PE induced ERK activation. Additionally, PLD1 activity was essential to PE induced stress fiber formation. In these studies, transfection with DN PLD2 had little effect on stress fiber formation, while DN PLD1 abrogated PE induced stress fibers. Subsequent use of a wounding assay to study cell migration identified that PE enhanced cell migration and that this enhancement was specifically blocked by DN PLD1. Our data shows that PLD1 and not PLD2 mediates α1-adrenergic regulation of cell migratory events and defines a unique role for PLD1 in signal transduction. Work supported by NIH Award number 1 R15 HL074924-01A1.