An analysis of the Populus root transcriptome was carried out in order to improve our understanding of molecular mechanisms underlying the response of trees to copper (Cu) stress. In a first stage, cuttings of nine Populus species were grown hydroponically and screened for differential phenotypical response to Cu. In a second stage, plants of the most Cu tolerant clone (Populus deltoides) were exposed to 30 and 60_MCuSO4 for 12 and 24 h. RNA isolated from root tissues was analyzed with 4.6 k cDNA arrays in order to determine alterations in transcription. Copper exposures increased significantly Cu concentration in roots. A series of transcripts differentially accumulated in a general or a specific way under the different stress conditions. Most of the transcripts encode proteins related to defense against biotic stress, antioxidant mechanisms, metal homeostasis, and water flux control. Expression of several transcripts involved in signaling, such as MAP kinases, calcium and ethylene-related enzymes were also altered under Cu exposure. Our results suggest the Cu accumulation in roots and a general defensive response against oxidative stress.