Nitrogen concentrations of C₃ species usually decline after prolonged exposure to elevated CO₂. This may result from a dependence of nitrate (NO₃^-) assimilation on photorespiration. To investigate the relationship between these two processes, we assessed NO₃^- assimilation under modified atmospheres using ¹⁵N techniques. Wheat (Triticum aestivum cv. Veery) seedlings at a photosynthetic flux density of 1000 μmol m^{‑2  sec-1} were exposed to an ambient atmosphere (360 μmol mol^-1 CO₂; 21% O₂), elevated CO₂ (720 μmol mol^-1 CO₂; 21 % O₂), or low O₂ (360 μmol mol^-1 CO₂; 2% O₂), and provided with nitrogen as ¹⁵N-enriched NO₃^- during a 12 h uptake period from a nutrient solution. Compared to ambient conditions, shoot nitrate assimilation rates decreased by 20% and 30% under elevated CO₂ and low O₂, respectively. In an alternative approach, we analyzed the d¹⁵N signature of NO₃^- and total N of wheat seedlings exposed to natural abundance levels of ¹⁴N and ¹⁵N. Given that the enzyme nitrate reductase discriminates strongly against the heavy isotope, the isotopic signature of organic N indicates the extent to which NO₃^- had become limited. In the shoots, d¹⁵N of organic N was lower, and higher for NO₃^- , in plants grown under elevated CO₂ compared to ambient conditions, indicating that NO₃^- availability at the site of reduction was more limited under ambient than elevated CO₂ concentrations. Both methods thus show that conditions diminishing photorespiration also suppress NO₃^- assimilation.