Phospholipase D (PLD) activity has been implicated in several aspects of cell physiology including vesicle transport, signal transduction, cell proliferation. cytoskeletal structure, and oncogenic transformation. Two PLD isoforms (PLD1 and PLD2) have been identified and characterized. We have expressed both wild-type and catalytically inactive forms of PLD I and PLD2 in 3Y1 rat fibroblasts and in 3Y1 cells transformed by v-Src, a tyrosine kinase that elevates PLD activity. The v-Src-transformed 3Y1 cells have small, but distinct cell protrusions, implicated in cell migration and metastasis. We report here that elevated expression of PLD2 substantially increased the length of the cell protrusions and that a catalytically inactive PLD2 mutant abolished the cell protrusions. The extended protrusions in the PLD2-overexpressing cells were dependent upon microtubule assembly. These data suggest a role for PLD2 in the v-Src-mediated formation of cell protrusions that may be critical for the invasive properties of v-Src-transformed cells. (C) 2002 Elsevier Science (USA). All rights reserved.