Experiments were carried out to clarify problems encountered in measuring metabolic and storage pool sizes of nitrate in wheat leaf sections with an in vivo nitrate reductase assay. The leaf sections were from seedlings grown on 15 millilar nitrate. Data obtained show that the cessation of nitrite accumulation, used as an index of the active nitrate pool size, could be caused by lack of anaerobiosis in the assay system, the lack of energy for nitrate reduction, or a loss of nitrate reductase activity. Availability of nitrate was never the limiting factor in this system. It is concluded that pool sizes of nitrate cannot be determined in wheat leaves with the in vivo assays employed. In 1973, Ferrari et al. (6) proposed that the in vivo assay for NR,3 with slight modifications, could be used to estimate the size of a metabolic (active) pool and a storage pool of nitrate in plant cells and tissues. Subsequently, other workers (2, 3) used this procedure to measure pool sizes in plant organs. Because NR is substrate-inducible, the pool sizes should affect the level of the enzyme and be a useful adjunct in attempts to use measurements of NRA in identifying crop cultivars with high production potential (7).The original objective of the work was to measure NRA and nitrate pool sizes in the leaves of growth chamber-grown seedlings of Australian wheats of commercial importance and to compare these values with their known field yield performance. Because our attempts to measure pool sizes with the in vivo method of Ferrari et al. (6) were unsuccessful, identification of the cause of this problem was attempted. These investigations led to a series of observations and conclusions concerning the proposed method for measuring pool sizes and factors that affect the in vivo assay. MATERIALS AND METHODS PLANT MATERIALIn Australia, seedlings of Triticum aestivum L., var. Olympic, were grown as described (19 Method A. Leaf sections (0.2 g) were added to vials or flasks (20-to 30-ml capacity) containing 10 ml 2 mm CaSO4. An initial volume of 8 ml CaSO4 was used when 2.0 ml of nitrate, malate etc. were to be added during the assay. All media were purged with N2 for 10 min prior to adding to the assay vials. N2 was bubbled through the medium of each vial for 3 min after the leaf sections had been added and just prior to sealing the vials with a rubber stopper. The samples were repurged with N2 for 3 min after vials were opened to supply additives. Incubation was in the dark at 30 C. Reactions were stopped at the desired time by transferring the vials (rubber stopper replaced with a marble or serum cap) to boiling water for 5 min. After cooling, aliquots of the liquid were removed for determination of nitrite (18). The CaS04 was used instead of phosphate because Ca2+ should help maintain membrane integrity.Method A-1 was similar to method A in all details except that nitrite accumulation was followed by sequential removal of aliquots of the medium at hourly intervals from the same assay vial, and after each sampling the conten...