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 per thousand 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.