The phospholipase D (PLD) family of Arabidopsis thaliana has 12 identified members, including three highly homologous PLDgammas. The enzymatic and molecular properties of PLDgamma2 were characterized and compared with those of PLDgamma1. Two variants of PLDgamma2 cDNAs, designated PLDgamma2a and PLDgamma2b, were isolated, and they differ in the presence of a 96-nucleotide fragment at the beginning of the open reading frame. Catalytically active PLDgamma2a was expressed in E. coli. PLDgamma2a and gamma1 both require phosphatidylinositol 4,5-bisphosphate (PIP(2)) and calcium for activity, but they differ in the effect of PIP(2) and Triton X-100 on their activities. While Triton X-100 could greatly activate PLDgamma1 activity and served only as a neutral diluent in the substrate vesicles, it totally abolished PLDgamma2a activity and prohibited any stimulation effect from PIP(2.) PLDgamma2a misses one of the basic, PIP(2)-interacting residues, which may weaken the binding of PIP(2) to PLDgamma2a. In addition, PLDgamma2 and PLDgamma1 displayed different patterns of expression in different tissues, and the transcript of PLDgamma2a differs from that of PLDgamma1 by having a longer 5'-UTR. These differences in biochemical and molecular properties suggest that the highly homologous PLDgammas are subjected to unique regulations and might have distinguishable functions.