AbstractMammalian phospholipase D (PLD), a multifunctional signaling enzyme, has been reported to facilitate neurite outgrowth in cultured neurons. However, two mammalian isoforms have been found, PLD1 and PLD2, and it has not been determined which isoform is involved, or whether this in vitro phenomenon is relevant to neurite extension in vivo. Using confocal microscopy, we demonstrate that the PLDs are expressed by different cell types in the mouse brain: PLD1 by neurons, and PLD2 by astrocytes. Moreover, using a model of experimentally induced hippocampal mossy fiber sprouting, both isoforms were observed to increase dramatically in expression level along tracts of mossy fiber spouting, supporting the proposal that PLD plays a role in this process. Given that the two isoforms undertake unique molecular functions in cultured cells, our findings suggest that in vivo PLD1 and PLD2 may modulate neuronal plasticity via different pathways and cell types. © 2004 Wiley‐Liss, Inc.