Assimilatory nitrate reductase activity (NRA) in crude spinach leaf (Spinacia oleracea) extracts undergoes rapid changes following fluctuations in photosynthesis brought about by changes in external CO(2) or by water stress (WM Kaiser, E Brendle-Behnisch [1991] Plant Physiol 96:363-367). A modulation of NRA sharing several characteristics (stability, response to Mg(2+) or Ca(2+), kinetic constants) with the in vivo modulation was obtained in vitro by preincubating desalted leaf extracts with physiological concentrations of Mg(2+) and ATP (deactivating) or AMP (activating). When nitrate reductase (NR) was inactivated in vivo by illuminating leaves at the CO(2) compensation point, it could be reactivated in vitro by incubating leaf extracts with AMP. For the in vitro inactivation, ATP could be replaced by GTP or UTP. Nonhydrolyzable ATP analogs (beta, gamma-imido ATP, beta, gamma-methyl-ATP) had no effect on NR, whereas gamma-S-ATP caused an irreversible inactivation. This suggests that NR modulation involves ATP hydrolysis. In contrast to NR in crude leaf extracts, partially purified NR did not respond to ATP or AMP. ATP and AMP levels in whole leaf extracts changed in the way predicted by the modulation of NRA when leaves were transferred from photosynthesizing (low ATP/AMP) to photorespiratory (high ATP/AMP) conditions. Adenine nucleotide levels in leaves could be effectively manipulated by feeding mannose through the leaf petiole. NRA followed these changes as expected from the in vitro results. This suggests that cytosolic ATP/AMP levels are indeed the central link between NRA in the cytosol and photosynthesis in the chloroplast. Phosphorylation/dephosphorylation of NR or of NR-regulating protein factors is discussed as a mechanism for a reversible modulation of NR by ATP and AMP.