Abstract Several different cellular processes determine the size of the metabolically available nitrate pool in the cytoplasm. These processes include not only ion fluxes across the plasma membrane and tonoplast but also assimilation by the activity of nitrate reductase (NR). In roots, the maintenance of cytosolic nitrate activity during periods of nitrate starvation and resupply (M. van der Leij, S.J. Smith, A.J. Miller [1998] Planta 205: 64–72; R.-G. Zhen, H.-W. Koyro, R.A. Leigh, A.D. Tomos, A.J. Miller [1991] Planta 185: 356–361) suggests that this pool is regulated. Under nitrate-replete conditions vacuolar nitrate is a membrane-bound store that can release nitrate to the cytoplasm; after depletion of cytosolic nitrate, tonoplast transporters would serve to restore this pool. To study the role of assimilation, specifically the activity of NR in regulating the size of the cytosolic nitrate pool, we have compared wild-type and mutant plants. In leaf mesophyll cells, light-to-dark transitions increase cytosolic nitrate activity (1.5–2.8 mm), and these changes were reversed by dark-to-light transitions. Such changes were not observed in nia1nia2 NR-deficient plants indicating that this change in cytosolic nitrate activity was dependent on the presence of functional NR. Furthermore, in the dark, the steady-state cytosolic nitrate activities were not statistically different between the two types of plant, indicating that NR has little role in determining resting levels of nitrate. Epidermal cells of both wild type and NR mutants had cytosolic nitrate activities that were not significantly different from mesophyll cells in the dark and were unaltered by dark-to-light transitions. We propose that the NR-dependent changes in cytosolic nitrate provide a cellular mechanism for the diurnal changes in vacuolar nitrate storage, and the results are discussed in terms of the possible signaling role of cytosolic nitrate.