Brassinosteroids (BRs) have been shown to induce hydrogen peroxide (H(2)O(2)) accumulation, and BR-induced H(2)O(2) up-regulates antioxidant defence systems in plants. However, the mechanisms by which BR-induced H(2)O(2) regulates antioxidant defence systems in plants remain to be determined. In the present study, the role of ZmMPK5, a mitogen-activated protein kinase, in BR-induced anitioxidant defence and the relationship between the activation of ZmMPK5 and H(2)O(2) production in BR signalling were investigated in leaves of maize (Zea mays) plants. BR treatment activated ZmMPK5, induced apoplastic and chloroplastic H(2)O(2) accumulation, and enhanced the total activities of antioxidant enzymes. Such enhancements were blocked by pre-treatment with mitogen-activated protein kinase kinase (MAPKK) inhibitors and H(2)O(2) inhibitors or scavengers. Pre-treatment with MAPKK inhibitors substantially arrested the BR-induced apoplastic H(2)O(2) production after 6 h of BR treatment, but did not affect the levels of apoplastic H(2)O(2) within 1 h of BR treatment. BR-induced gene expression of NADPH oxidase was also blocked by pre-treatment with MAPKK inhibitors and an apoplastic H(2)O(2) inhibitor or scavenger after 120 min of BR treatment, but was not affected within 30 min of BR treatment. These results suggest that the BR-induced initial apoplastic H(2)O(2) production activates ZmMPK5, which is involved in self-propagation of apoplastic H(2)O(2) via regulation of NADPH oxidase gene expression in BR-induced antioxidant defence systems.