Three phospholipase Ds (PLDs; EC 3.1.4.4) have been cloned from Arabidopsis, and they exhibit two distinct types of activities: polyphosphoinositide-requiring PLDbeta and PLDgamma, and polyphosphoinositide-independent PLDalpha. In subcellular fractions of Arabidopsis leaves, PLDalpha and PLDgamma were both present in the plasma membrane, intracellular membranes, mitochondria, and clathrin-coated vesicles, but their relative levels differed in these fractions. In addition, PLDgamma was detected in the nuclear fraction. In contrast, PLDbeta was not detectable in any of the subcellular fractions. PLDalpha activity was higher in the metabolically more active organs such as flowers, siliques, and roots than in dry seeds and mature leaves, whereas the polyphosphoinositide-dependent PLD activity was greater in older, senescing leaves than in other organs. PLDbeta mRNA accumulated at a lower level than the PLDalpha and PLDgamma transcripts in most organs, and the expression pattern of the PLDbeta mRNA also differed from that of PLDalpha and PLDgamma in different organs. Collectively, these data demonstrated that PLDalpha, PLDbeta, and PLDgamma have different patterns of subcellular distribution and tissue expression in Arabidopsis. The present study also provides evidence for the presence of an additional PLD that is structurally more closely related to PLDgamma than to the other two PLDs.