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High ozone (O-3) pollution impairs the carbon and water balance of trees, which is of special interest in planted forests. However, the effect of long-term O-3 exposure on tree growth and water use, little remains known. In this study, we analysed the relationships of intra-annual stem growth pattern, seasonal sap flow dynamics and xylem morphology to assess the effect of long term O-3 exposure of mature O-3-sensitive hybrid poplars (Oxford' clone). Rooted cuttings were planted in autumn 2007 and drip irrigated with 2 liters of water as ambient O-3 treatment, or 450 ppm ethylenediurea (N-[2-(2-oxo-1-imidazolidinyl)ethyl]-N0-phenylurea, abbreviated as EDU) solution as O-3 protection treatment over all growing seasons. During 2013, point dendrometers and heat pulses were installed to monitor radial growth, stem water relations and sap flow. Ambient O-3 did not affect growth rates, even if the seasonal culmination point was 20 days earlier on average than that recorded in the O-3 protected trees. Under ambient O-3, trees showed reduced seasonal sap flow, however, the lower water use was due to a decrease of Huber value (decrease of leaf area for sapwood unit) rather than to a change in xylem morphology or due to a direct effect of sluggish stomatal responses on transpiration. Under high evaporative demand and ambient O-3 concentrations, trees showed a high use of internal stem water resources modulated by stomatal sluggishness, thus predisposing them to be more sensitive water deficit during summer. The results of this study help untangle the compensatory mechanisms involved in the acclimation processes of forest species to long-term O-3 exposure in a context of global change.