Maize (Zea mays L.) grown on low (0.8 raM) NO;-, as well as untransformed and transformed Nicotiana plumbaginifolia constitutively expressing nitrate reductase (NR), was used to study the effects of NO~ on the NR activation state. The NR activation state was determined from the relationship of total activity extracted in the presence of ethylenediaminetetracetic acid to that extracted in the presence of Mg 2+. Light activation was observed in both maize and tobacco leaves. In the tobacco lines, NO3 did not influence the NR activation state. In excised maize leaves, no correlation was found between the foliar NO~ content and the NR activation state. Similarly, the NR activation state did not respond to NOj. Since the NR activation state determined from the degree of Mg 2 +-induced inhibition of NR activity is considered to reflect the phosphorylation state of the NR protein, the protein phosphatase inhibitor microcystin LR was used to test the importance of protein phosphorylation in the NO~-induced changes in NR activity. In-vivo inhibition of endogenous protein phosphatase activity by microcystin-LR decreased the level of NR activation in the light. This occurred to the same extent in the presence or absence of exogenous NO3. We conclude that NO3 does not effect the NR activation state, as modulated by protein phosphorylation in either tobacco (a C3 species) or maize (a C4 species). The short-term regulation of NR therefore differs from the NO~-mediated responses observed for phosphoenolpyruvate carboxylase and sucrose phosphate synthase.