An open-top chamber experiment was carried out in Curno (Northern Italy) in 2004 and 2005 on seedlings of Fagus sylvatica (FS), Quercus robur (QR), and an ozone-sensitive Populus (POP) clone, to investigate the role of two stress factors: tropospheric ozone and water shortage. Treatments were filtered air to achieve a 50% reduction in the environmental ozone concentrations (charcoal filtered, CF); and non-filtered air, with a 5% reduction in the environmental ozone concentrations (non-filtered, NF). Overall ozone exposure (AOT40) in open air (April–September) was 26,995 ppb h in 2004 and 25,166 ppb h in 2005. The plants were either watered (W) or not watered (dry, D). We investigated the above-ground biomass, tree-ring growth, stable carbon isotopes ratio, i.e. d13C of tree rings, and the photosynthetic parameter Driving forces (DFABS), derived from chlorophyll a fluorescence analysis. Ozone-induced growth reduction (in terms of biomass) in POP, and that reduction was more pronounced in D plots. A synergistic effect of ozone and drought stress was evidenced by DFABS in POP and QR, but not in FS. The water availability was revealed as the main factor influencing the isotopic ratio d13C. In droughtstressed seedlings, the increase in d13C value was accompanied by the reduction in stomatal conductance and increased DFABS. Fast-growing plant species with high water requirements are more susceptible to ozone and drought stress.