This study was conducted to determine whether diurnal fluctuations of nitrate reductase activity in two cotton species, Pima (Gossypium barbadense L.) and Acala (Gossypium hirsutum L.), are regulated by NO−3 uptake or leaf NO−3 concentration. The two species differ greatly in their N use efficiency. The seedlings were grown for 25 d in nutrient solutions containing 0.05, 0.10 or 1.0 mM NO−3 under a 14‐h light/10‐h dark cycle at 30°C/20°C. Uptake rates were measured by following NO−3 depletion from the uptake solutions at 20°C or 30°C. Nitrate reductase activity (NRA) in fully expanded first true leaves was determined by an anaerobic method in vivo. Upon illumination uptake rates for both species, measured at 30°C, increased until they plateaued in about 5 h and were maintained at that level for the remainder of the light period and the following dark cycle. When measured at 20°C in the dark, however, the uptake rates decreased 25 to 30% for the first 3 h and then remained constant. Leaf NRA increased rapidly during the first hour of illumination, reached a plateau and then decreased after 4 to 6 h of illumination. The decline was more rapid when NRA was assayed in the absence of NO−3 In darkness, NRA levels were low and did not fluctuate. Upon illumination, leaf NO−3 concentration also increased for about 7 h and then decreased gradually. The results indicate that (i) the diurnal rhythm of NO−3 uptake is modulated by temperature rather than by light/dark transitions, and (ii) while the increase in NRA upon illumination may be regulated by NO−3 flux from roots to shoots, the decrease in NRA under prolonged illumination is independent of that flux.