We analyzed ¹⁴C, ¹³C and ¹⁸O isotope variations over a 50-year period in tree rings of Quercus ilex L. trees growing at a natural CO₂ spring in a Mediterranean ecosystem. We compared trees from two sites, one with high and one with low exposure to CO₂ from the spring. The spring CO₂ is free of ¹⁴C. Thus, this carbon can be traced in the wood, and the amount originating from the spring calculated. The amount decreased over time, from about 40% in 1950 to 15% at present for the site near the spring, indicating a potential difficulty in the use of natural CO₂ springs for elevated CO₂ research. The reason for the decrease may be decreasing emission from the spring or changes in stand structure, e.g., growth of the canopy into regions with lower concentrations. We used the ¹⁴C-calculated CO₂ concentration in the canopy to determine the ¹³C discrimination of the plants growing under elevated CO₂ by calculating the effective canopy air ¹³C/¹²C isotopic composition. The trees near the spring showed a 2.5‰ larger ¹³C discrimination than the more distant trees at the beginning of the investigated period, i.e., for the young trees, but this difference gradually disappeared. Higher discrimination under elevated CO₂ indicated reduced photosynthetic capacity or increased stomatal conductance. The latter assumption is unlikely as inferred from the ¹⁸O data, which were insensitive to CO₂ concentration. In conclusion, we found evidence for a downward adjustment of photosynthesis under elevated CO₂ in Q. ilex in this dry, nutrient-poor environment.