Background/Question/Methods Vegetation is an important sink for nitrogen deposition. In nitrogen-limited ecosystems, atmospheric reactive nitrogen compounds may represent a considerable source of nitrogen to vegetation and have an impact on plant metabolism. However, few studies have investigated the magnitude of leaf uptake of gaseous nitrogen pollution separate from soil-deposited nitrogen at realistic concentrations. Further, the mechanisms driving differences in leaf nitrogen uptake capacity among plant species are still unresolved. Nitrogen isotope ratios (¹⁵N:¹⁴N) are useful indicators for exploring the leaf uptake of gaseous nitrogen because nitrogen sources with unique isotopic values can be used to partition the relative strength of each source. In this study, we conducted an enriched ¹⁵N experiment in a custom-built hydroponics-fumigation system where varying amounts of reactive nitrogen were supplied to the leaves (as ¹⁵NO₂-enriched air) or to the roots [as nitrate (NO₃^-) or ammonium (NH₄⁺)]. The objective of this study was to examine the influence of different root N sources (NO₃^- vs. NH₄⁺) and associated nitrogen assimilatory pathways on patterns of leaf uptake of nitrogen in tobacco (Nicotiana tabacum). Specifically, we examined differences in growth, leaf δ¹⁵N values, nitrate reductase (NR) activity, and glutamate synthetase (GS) activity to explore leaf NO₂ uptake capacity. Results/Conclusions Total biomass accumulation was higher in plants exposed to high levels of root N supply (both NO₃^- and NH₄⁺) regardless of NO₂ exposure. NO₂-derived nitrogen in leaf tissue varied between 7 and 20%, with leaf NO₂ uptake highest in plants supplied with low levels of NO₃^-. Leaf NO₂ exposure increased both leaf NR and GS activities, indicating a relationship between leaf uptake capacity and the activity in the primary nitrogen assimilation pathways through which NO₂ is metabolized. Root GS activity was higher in plants supplied with NH₄⁺, while root NR activity was higher in plants supplied with NO₃^- as the root N source. Results indicate that biomass production was independent of the nitrogen source present in the nutrient solution, but very much influenced by nitrogen concentration (increasing either NO₃^- or NH₄⁺ concentration led to higher biomass accumulation). Further, enzyme expression of the primary nitrogen metabolic pathways appears to be a potentially significant factor controlling leaf uptake capacity, patterns in leaf δ¹⁵N values, and variation in N assimilation among species. In conclusion, this study contributes to the mechanistic understanding of the differences in direct leaf uptake capacity among species by integrating relationships among assimilatory enzyme activity and sources of available nitrogen.