AbstractIt is uncertain how the predicted further rise of atmospheric carbon dioxide (CO₂) concentration will affect plant nutrient availability in the future through indirect effects on the gross rates of nitrogen (N) mineralization (production of ammonium) and depolymerization (production of free amino acids) in soil. The response of soil nutrient availability to increasing atmospheric CO₂ is particularly important for nutrient poor ecosystems. Within a FACE (Free-Air Carbon dioxide Enrichment) experiment in a native, nutrient poor Eucalyptus woodland (EucFACE) with low soil organic matter (≤ 3%), our results suggested there was no shortage of N. Despite this, microbial N use efficiency was high (c. 90%). The free amino acid (FAA) pool had a fast turnover time (4 h) compared to that of ammonium (NH₄⁺) which was 11 h. Both NH₄-N and FAA-N were important N pools; however, protein depolymerization rate was three times faster than gross N mineralization rates, indicating that organic N is directly important in the internal ecosystem N cycle. Hence, the depolymerization was the major provider of plant available N, while the gross N mineralization rate was the constraining factor for inorganic N. After two years of elevated CO₂, no major effects on the pools and rates of the soil N cycle were found in spring (November) or at the end of summer (March). The limited response of N pools or N transformation rates to elevated CO₂ suggest that N availability was not the limiting factor behind the lack of plant growth response to elevated CO₂, previously observed at the site.