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Abstract: The phospholipase D (PLD) family of Arabidopsis thaliana has 12 identified members, including three highly homologous PLDγs. The enzymatic and molecular properties of PLDγ2 were characterized and compared with those of PLDγ1. Two variants of PLDγ2 cDNAs, designated PLDγ2a and PLDγ2b, were isolated, and they differ in the presence of a 96-nucleotide fragment at the beginning of the open reading frame. Catalytically active PLDγ2a was expressed in E. coli. PLDγ2a and γ1 both require phosphatidylinositol 4,5-bisphosphate (PIP2) and calcium for activity, but they differ in the effect of PIP2 and Triton X-100 on their activities. While Triton X-100 could greatly activate PLDγ1 activity and served only as a neutral diluent in the substrate vesicles, it totally abolished PLDγ2a activity and prohibited any stimulation effect from PIP2. PLDγ2a misses one of the basic, PIP2-interacting residues, which may weaken the binding of PIP2 to PLDγ2a. In addition, PLDγ2 and PLDγ1 displayed different patterns of expression in different tissues, and the transcript of PLDγ2a differs from that of PLDγ1 by having a longer 5′-UTR. These differences in biochemical and molecular properties suggest that the highly homologous PLDγs are subjected to unique regulations and might have distinguishable functions. [Copyright &y& Elsevier]