In order to functionally analyze the predicted defensive role of leaf polyphenol oxidase (PPO; EC 18.104.22.168) in Populus, transgenic hybrid aspen (Populus tremula x P. alba) plants overexpressing a hybrid poplar (Populus trichocarpa x P. deltoides) PtdPPO1 gene were constructed. Regenerated transgenic plants showed high PPO enzyme activity, PtdPPO1 mRNA levels and PPO protein accumulation. In leaf disk bioassays, forest tent caterpillar (Malacosoma disstria) larvae feeding on PPO-overexpressing transgenics experienced significantly higher mortality and reduced average weight gain compared to larvae feeding on control leaves. However, this effect was observed only when older egg masses were used and the resulting larvae showed reduced growth and vigor. In choice tests, no effect of PPO overexpression was detected. Although PPO in poplar leaves is latent and requires activation with detergents or trypsin for full enzymatic activity, in caterpillar frass the enzyme was extracted in the fully activated form. This activation correlated with partial proteolytic cleavage, suggesting that PPO latency and activation during digestion could be an adaptive and defense-related feature of poplar PPO.