Elevated expression of phospholipase D (PLD) in rat fibroblasts overexpressing a tyrosine kinase leads to cell transformation. However, it has been difficult to get elevated expression of PLD in normal rat fibroblasts. Using transient transfection and an inducible expression system, we were able to get elevated expression of PLD1 and PLD2 in 3Y1 rat fibroblasts. Elevated expression of either PLD1 or PLD2 in 3Y1 cells led to apoptosis in the absence of serum. Elevated PLD expression resulted in reduced cell viability and the cleavage of the caspase 3 substrates poly-ADP-ribose polymerase (PARP) and protein kinase C delta. Elevated PLD expression also stimulated cytochrome c release, indicating that the mitochondrial apoptosis pathway was activated. Thus, while elevated PLD expression can transform cells with elevated tyrosine kinase expression, elevated expression of PLD activity in normal cells renders cells sensitive to apoptotic insult. (C) 2002 Elsevier Science (USA). All rights reserved.