Abstract: The mechanisms of H₂O₂ generation in guard cells of Vicia faba induced by abscisic acid (ABA) were measured by using fluorescene probe, 8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt (HPTS). When guard cells were treated with ABA, fluorescene quenching of HPTS occurred, and the treatment of the guard cells with diphenyleneiodonium(DPI) at 10^-6 mol/mL (final concentration) which is known as an inhibitor of supero xide generating NADPH oxidases nearly reversed the quenching reaction induced by ABA O₂ mmol/L(final concentration). ABA and H₂O₂ might induce stomatal closure. Furthermore, when DPI or catalase(CAT)(10³ unit/mL) was added to the suspension after above treatment reversed ABA-induced stomatal closure. These results suggest that a novel O₂  generating NADPH oxidase in the plasma membrane of guard cell of Vicia faba may be activated by ABA, and subsequently H₂O₂, which involved in the signal transduction pathway of ABA-induced stomatal closure.