A novel plant phospholipase D (PLD; EC 22.214.171.124) activity, which is dependent on phosphatidylinositol 4,5-bisphosphate (PIP2) and nanomolar concentrations of calcium, has been identified in Arabidopsis. This report describes the cloning, expression, and characterization of an Arabidopsis cDNA that encodes this PLD. We have designated names of PLDβ for this PIP2- dependent PLD and PLDα for the previously characterized PIP2-independent PLD that requires millimolar Ca2+ for optimal activity. The PLDβ cDNA contains an open reading frame of 2904 nucleotides coding for a 968-amino acid protein of 108,575 daltons. Expression of this PLDβ cDNA clone in Escherichia coli results in the accumulation of a functional PLD having PLDβ, but not PLDα, activity. The activity of the expressed PLDβ is dependent on PIP2 and submicromolar amounts of Ca2+, inhibited by neomycin, and stimulated by a soluble factor from plant extracts. Sequence analysis reveals that PLDβ is evolutionarily divergent from PLDα and that its N terminus contains a regulatory Ca2+-dependent phospholipid-binding (C2) domain that is found in a number of signal transducing and membrane trafficking proteins.