In response to herbivore (Spodoptera littoralis) attack, lima bean (Phaseolus lunatus) leaves produced hydrogen peroxide (H(2)O(2)) in concentrations that were higher when compared to mechanically damaged (MD) leaves. Cellular and subcellular localization analyses revealed that H(2)O(2) was mainly localized in MD and herbivore-wounded (HW) zones and spread throughout the veins and tissues. Preferentially, H(2)O(2) was found in cell walls of spongy and mesophyll cells facing intercellular spaces, even though confocal laser scanning microscopy analyses also revealed the presence of H(2)O(2) in mitochondria/peroxisomes. Increased gene and enzyme activations of superoxide dismutase after HW were in agreement with confocal laser scanning microscopy data. After MD, additional application of H(2)O(2) prompted a transient transmembrane potential (V(m)) depolarization, with a V(m) depolarization rate that was higher when compared to HW leaves. In transgenic soybean (Glycine max) suspension cells expressing the Ca(2+)-sensing aequorin system, increasing amounts of added H(2)O(2) correlated with a higher cytosolic calcium ([Ca(2+)](cyt)) concentration. In MD and HW leaves, H(2)O(2) also triggered the increase of [Ca(2+)](cyt), but MD-elicited [Ca(2+)](cyt) increase was more pronounced when compared to HW leaves after addition of exogenous H(2)O(2). The results clearly indicate that V(m) depolarization caused by HW makes the membrane potential more positive and reduces the ability of lima bean leaves to react to signaling molecules.