H(2)O(2) is an essential signal in absicic acid (ABA)-induced stomatal closure. It can be synthesized by several enzymes in plants. In this study, the roles of copper amine oxidase (CuAO) in H(2)O(2) production and stomatal closure were investigated. Exogenous ABA stimulated apoplast CuAO activity, increased H(2)O(2) production and [Ca(2+)](cyt) levels in Vicia faba guard cells, and induced stomatal closure. These processes were impaired by CuAO inhibitor(s). In the metabolized products of CuAO, only H(2)O(2) could induce stomatal closure. By the analysis of enzyme kinetics and polyamine contents in leaves, putrescine was regarded as a substrate of CuAO. Putrescine showed similar effects with ABA on the regulation of H(2)O(2) production, [Ca(2+)](cyt) levels, as well as stomatal closure. The results suggest that CuAO in V. faba guard cells is an essential enzymatic source for H(2)O(2) production in ABA-induced stomatal closure via the degradation of putrescine. Calcium messenger is an important intermediate in this process.