Growth chamber studies with soybeans (Glycine max [L.] Merr.) were designed to determine the relative limitations of NO3−, NADH, and nitrate reductase (NR) per se on nitrate metabolism as affected by light and temperature. Three NR enzyme assays (+NO3−in vivo, −NO3−in vivo, and in vitro) were compared. NR activity decreased with all assays when plants were exposed to dark. Addition of NO3− to the in vivo NR assay medium increased activity (over that of the −NO3−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 NO3− was rate-limiting. The stimulation of in vivo NR activity by NO3− was not seen in plants exposed to extended dark periods at elevated temperatures (16 hr-30 C), indicating that under those conditions, NO3− was not the limiting factor. Under the latter condition, in vitro NR activity was appreciable (19 μmol NO2− [g fresh weight, hr]−1) suggesting that enzyme level per se was not the limiting factor and that reductant energy might be limiting.