Abstract Four types of phospholipase D (PLD), PLDα, β, γ, and δ, have been characterized in Arabidopsis, and they display different requirements for Ca2+, phosphatidylinositol 4,5-bisphosphate (PIP2), substrate vesicle composition, and/or free fatty acids. However, all previously cloned plant PLDs contain a Ca2+-dependent phospholipid-binding C2 domain and require Ca2+ for activity. This study documents a new type of PLD, PLDζ1, which is distinctively different from previously characterized PLDs. It contains at the N terminus a Phox homology domain and a pleckstrin homology domain, but not the C2 domain. A full-length cDNA for Arabidopsis PLDζ1 has been identified and used to express catalytically active PLD in Escherichia coli. PLDζ1 does not require Ca2+ or any other divalent cation for activity. In addition, it selectively hydrolyzes phosphatidylcholine, whereas the other Arabidopsis PLDs use several phospholipids as substrates. PLDζ1 requires PIP2 for activity, but unlike the PIP2-requiring PLDβ or γ, phosphatidylethanolamine is not needed in substrate vesicles. These differences are described, together with a genomic analysis of 12 putative Arabidopsis PLD genes that are grouped into α, β, δ, γ, and ζ based on their gene architectures, sequence similarities, domain structures, and biochemical properties.