This study was conducted to determine whether the inhibition of nitrate reductase activity (NRA; EC 1.6.6.1) in barley ( Hordeum vulgare L. var. CM-72) roots by the amino acids (glutamic, aspartic, glutamine and asparagine) is a direct effect or indirect due to inhibition of the NO 3 − uptake system. Roots of 8-day-old intact seedlings were supplied with the amino acids (I mM) individually either with NO 3 − (0.1 or 10 mM) or roots were pretreated with the amino acids and then supplied with NO 3 − only. Nitrate uptake was determined by following NO 3 − depletion from the uptake solution containing 0.1 mM NO 3 −. All the amino acids inhibited the increase in NO 3 − uptake similarly (50–60%) when the roots were supplied with 0.1 mM NO 3 −. Pretreatment with glutamic and aspartic acids was more inhibitory (70–80%) than with glutamine and asparagine (30%). The amino acids partially inhibited (35%) the induction of NRA in roots supplied with 0.1 mM NO 3 −; however, no inhibition occurred at 10 mM NO 3 −. Likewise, pretreatment with glutamic or aspartic acid inhibited the induction of NRA at 0.1 mM NO 3 − but not at 10 mM NO 3 −. In contrast, pretreatment with glutamine or asparagine had no effect on the subsequent induction of NRA, even at 0.1 mM NO 3 −. The results suggest that, at low NO 3 − supply, the inhibition of induction of NRA by the amino acids is a result of the lack of substrate availability due to inhibition of the NO 3 − uptake system.