The phytoalexin deficient 4 (PAD4) gene in Arabidopsis thaliana (AtPAD4) is involved in the regulation of plant--pathogen interactions. The role of PAD4 in woody plants is not known; therefore, we characterized its function in hybrid aspen and its role in reactive oxygen species (ROS)-dependent signalling and wood development. Three independent transgenic lines with different suppression levels of poplar PAD expression were generated. All these lines displayed deregulated ROS metabolism, which was manifested by an increased H2O2 level in the leaves and shoots, and higher activities of manganese superoxide dismutase (MnSOD) and catalase (CAT) in the leaves in comparison to the wild-type plants. However, no changes in non-photochemical quenching (NPQ) between the transgenic lines and wild type were observed in the leaves. Moreover, changes in the ROS metabolism in the pad4 transgenic lines positively correlated with wood formation. A higher rate of cell division, decreased tracheid average size and numbers, and increased cell wall thickness were observed. The results presented here suggest that the Populus tremula × tremuloides PAD gene might be involved in the regulation of cellular ROS homeostasis and in the cell division--cell death balance that is associated with wood development.