Growth chamber studies with soybeans (Glycine max [L.] Merr.) were designed to determine the relative limitations of NO(3) (-), NADH, and nitrate reductase (NR) per se on nitrate metabolism as affected by light and temperature. Three NR enzyme assays (+NO(3) (-)in vivo, -NO(3) (-)in vivo, and in vitro) were compared. NR activity decreased with all assays when plants were exposed to dark. Addition of NO(3) (-) to the in vivo NR assay medium increased activity (over that of the -NO(3) (-)in vivo assay) at all sampling periods of a normal day-night sequence (14 hr-30 C day; 10 hr-20 C night), indicating that NO(3) (-) was rate-limiting. The stimulation of in vivo NR activity by NO(3) (-) was not seen in plants exposed to extended dark periods at elevated temperatures (16 hr-30 C), indicating that under those conditions, NO(3) (-) was not the limiting factor. Under the latter condition, in vitro NR activity was appreciable (19 mumol NO(2) (-) [g fresh weight, hr](-1)) suggesting that enzyme level per se was not the limiting factor and that reductant energy might be limiting.The addition of NADH to the in vivo NR assay medium did not stimulate NR activity, although it was not established that NADH entered the tissue. The addition of glucose, fructose 1,6-diphosphate, pyruvate, citrate, succinate, or malate to the in vivo assay medium significantly increased measurable NR activity of leaf tissue from plants pretreated to extended dark periods at elevated temperature. Glucose additions were most effective, usually stimulating increases 2- to 3-fold greater than the other metabolites. Increased NR activities from the various additives were attributed to production of NADH. The loss of in vivo NR activity in soybeans during darkness appeared to be due to the combination of a net loss of enzyme per se and energy depletion. The subsequent light stimulation of NR activity was likely due to increased availability of reductant energy as well as a net synthesis of the NR enzyme.