Phospholipase D (PLD) is expressed in many tissues and stimulated by growth factors and cytokines. However, the role of PLD in signal transduction is still not well-understood. Human embryonic kidney (HEK-293) cells exhibit low levels of both PLD1 and PLD2 mRNA, however, only PLD1 protein was detected by Western blot. When either isoform of PLD was stably expressed in HEK-293 cells, we observed an increased PLD activity in a cell-free system and a 12-O-tetradecanoyl-13-phorbol acetate (TPA)-stimulated increase in PLD activity in intact cells. This system was then used to elucidate the effects of PLD activity on TPA-stimulated signaling pathways. Two such pathways, the mitogen-activated protein kinases (MAPK), extracellular regulated protein kinase (ERK) and p38 are activated by growth factors and cellular stress, respectively. We found that TPA stimulated ERK phosphorylation regardless of the expression status of PLD. In contrast to ERK kinase, HEK-293 cells were unable to induce p38 phosphorylation by TPA stimulation. When HEK-293 cells expressed either PLD1 or PLD2, we observed elevated p38 phosphorylation in response to TPA stimulation. The ERK and p38 MAPKs can also stimulate the expression of both cyclooxygenase-2 (Cox-2) and interleukin-8 (IL-8). We used this system to differentiate the effect of PLD1 or PLD2 activity on the expression of Cox-2 and IL-8. Increased Cox-2 and IL-8 expression was found only in HEK-293 cells expressing PLD1. These data identify a novel role for the PLD1 isoform in the induction of gene expression and provide new insight into the differential role of PLD1 and PLD2 in cells.