Tropospheric ozone, one of the most phytotoxic air pollutants, may specially impose in long-lived forest trees substantial reduction in productivity and biomass. European beech saplings grown in lysimeter around areas were used to monitor proteomic changes upon elevated ozone concentrations following four vegetation periods of exposure. A proteome study based on highly sensitive two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) was performed to identify protein changes in European beech, the most important deciduous tree in Central Europe. Main emphasis was on identifying differentially expressed proteins after long-time period of ozone exposure under natural conditions rather than short-term responses or reactions under controlled conditions. Our results clearly demonstrate a response of European beech saplings to long-term ozone fumigation at the protein level. We indicate changes in the protein abundance of 142 protein spots; among them 59 were increased and 83 decreased following three years of elevated ozone exposure. As the first step, 40 proteins were identified by a homology driven mass spectrometric approach. Some of the identified proteins have been previously described in the context of short-term ozone responses in plants, indicating, at least for certain cellular functions, the congruence of plant reactions following short-and long-term ozone exposure. Under elevated ozone exposure, abundance of proteins related to the Calvin cycle and photosynthetic electron transport chain were decreased whereas the abundance of proteins regarding the carbon metabolism/catabolism were increased.