Leaf-level photosynthetic processes and their environmental dependencies are critical for estimating CO2 uptake from the atmosphere. These estimates use biochemical-based models of photosynthesis that require accurate Rubisco kinetics. We investigated the effects of canopy position, elevated atmospheric CO2 [eC; ambient CO2 (aC)+240 ppm] and elevated air temperature (eT; ambient temperature (aT)+3 °C) on Rubisco content and activity together with the relationship between leaf N andVcmax (maximal Rubisco carboxylation rate) of 7 m tall, soil-grownEucalyptus globulustrees. The kinetics ofE. globulusand tobacco Rubisco at 25 °C were similar.In vitroestimates ofVcmax derived from measures ofE. globulusRubisco content and kinetics were consistent, although slightly lower, than thein vivorates extrapolated from gas exchange. InE. globulus, the fraction of N invested in Rubisco was substantially lower than for crop species and varied with treatments. Photosynthetic acclimation ofE. globulusleaves to eC was underpinned by reduced leaf N and Rubisco contents; the opposite occurred in response to eT coinciding with growth resumption in spring. Our findings highlight the adaptive capacity of this key forest species to allocate leaf N flexibly to Rubisco and other photosynthetic proteins across differing canopy positions in response to future, warmer and elevated [CO2] climates.