The roles of the plasma-membrane (PM) NADPH oxidase in abscisic acid (ABA)- and water stress-induced antioxidant defense were investigated in leaves of maize ( Zea mays L.) seedlings. Treatment by exogenous ABA (100 micro M ABA) or osmotic stress (-0.7 MPa induced by polyethylene glycol) significantly increased the activity of the PM NADPH oxidase, the production of leaf O(2)(-), the activities of several antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase), and the contents of antioxidant metabolites (ascorbate and reduced glutathione). Pretreatment with three different inhibitors of NADPH oxidase (diphenylene iodonium, imidazole and pyridine) or an inhibitor of ABA biosynthesis (tungstate) reduced the increase in the activity of the PM NADPH oxidase and the production of leaf O(2)(-), and the capacity of antioxidant defense systems mediated by ABA. The inhibitory effects above caused by tungstate were reversed by exogenous ABA. These data indicate that NADPH oxidase is involved in the ABA-induced production of active oxygen species (AOS), and our results depict a minimal chain of events initiated by water stress-induced ABA accumulation, which then triggers the production of AOS by membrane-bound NADPH oxidase, resulting in the induction of antioxidant defense systems against oxidative damage in plants.