The effects of drought stress on phospholipase D (PLD) gene expression and enzymatic activity were investigated in ‘Pineapple’ sweet orange. PLDs are tissue-specific, with overlapping functions, and in response to stress they may interact with ABA signaling. Tissue specificity for expression of PLDs and their regulation are unknown in citrus. To assess PLD response to water stress and gene expression/regulation in citrus, we subjected potted seedlings to increasing levels of soil drought. Evapotranspirational demand (ET) was estimated by measuring weight loss in pots, and water stress was further assessed by measuring ABA content. Three treatments were performed over a 3-week period: (a) Control treatment without drought stress (100% of daily ET); (b) mild water stress (50% ET); and (c) severe water stress (0% ET). ABA content increased during drought stress in both roots and leaves, being higher in leaves than in roots by the end of the experiment for any stress condition assayed. PLD enzymatic activity was monitored and expression of five PLD genes was studied. PLD activity increased linearly over time in response to increased soil drought and was around three times higher in roots than in leaves. PLD activation occurred initially in roots and then in leaves. PLD gene expression in response to stress soil drought differed between roots and leaves. These results show the potential of PLD as a suitable indicator of stress severity in citrus.