The nitrogen isotope composition (δ15N) of plants has potential to provide time-integrated information on nitrogen uptake, assimilation and allocation. Here, we take advantage of existing T-DNA and γ-ray mutant lines of Arabidopsis thaliana to modify whole-plant and organ-level nitrogen isotope composition. Nitrate reductase 2 (nia2), nitrate reductase 1 (nia1) and nitrate transporter (nrt2) mutant lines and the Col-0 wild type were grown hydroponically under steady-state NO3– conditions at either 100 or 1000 μM NO3– for 35 days. There were no significant effects on whole-plant discrimination and growth in the assimilatory mutants (nia2 and nia1). Pronounced root vs leaf differences in δ15N, however, indicated that nia2 had an increased proportion of nitrogen assimilation of NO3– in leaves while nia1 had an increased proportion of assimilation in roots. These observations are consistent with reported ratios of nia1 and nia2 gene expression levels in leaves and roots. Greater whole-plant discrimination in nrt2 indicated an increase in efflux of unassimilated NO3– back to the rooting medium. This phenotype was associated with an overall reduction in NO3– uptake, assimilation and decreased partitioning of NO3– assimilation to the leaves, presumably because of decreased symplastic intercellular movement of NO3– in the root. Although the results were more varied than expected, they are interpretable within the context of expected mechanisms of whole-plant and organ-level nitrogen isotope discrimination that indicate variation in nitrogen fluxes, assimilation and allocation between lines.