Specific cellular components have been identified to function in abscisic acid (ABA) regulation of stomatal apertures, including calcium, the cytoskeleton, and phosphatidic acid. In this study, the regulation and dynamic organization of microtubules during ABA-induced stomatal closure by phospholipase D (PLD) and its product PA were investigated. ABA induced microtubule depolymerization and stomatal closure in wide-type (WT) Arabidopsis, whereas these processes were impaired in PLD mutant (pldα1). The microtubule-disrupting drugs oryzalin or propyzamide induced microtubule depolymerization, but did not affect the stomatal aperture, whereas their co-treatment with ABA resulted in stomatal closure in both WT and pldα1. In contrast, the microtubule-stabilizing drug paclitaxel arrested ABA-induced microtubule depolymerization and inhibited ABA-induced stomatal closure in both WT and pldα1. In pldα1, ABA-induced cytoplasmic Ca(2+) ([Ca(2+)]cyt) elevation was partially blocked, and exogenous Ca(2+)-induced microtubule depolymerization and stomatal closure were impaired. These results suggested that PLDα1 and PA regulate microtubular organization and Ca(2+) increases during ABA-induced stomatal closing and that crosstalk among signaling lipid, Ca(2+), and microtubules are essential for ABA signaling.