Nitrate is a major nitrogen (N) source for most crops. Nitrate uptake by root cells is a key step of nitrogen metabolism and has been widely studied at the physiological and molecular levels. Understanding how nitrate uptake is regulated will help us engineer crops with improved nitrate uptake efficiency. The present study investigated the regulation of the high-affinity nitrate transport system (HATS) by exogenous abscisic acid (ABA) and glutamine (Gln) in wheat (Triticum aestivum L.) roots. Wheat seedlings grown in nutrient solution containing 2 mmol/L nitrate as the only nitrogen source for 2 weeks were deprived of N for 4 d and were then transferred to nutrient solution containing 50 μmol/L ABA, and 1 mmol/L Gln in the presence or absence of 2 mmol/L nitrate for 0, 0.5, 1, 2, 4, and 8 h. Treated wheat plants were then divided into two groups. One group of plants was used to investigate the mRNA levels of the HATS components NRT2 and NAR2 genes in roots through semi-quantitative RT-PCR approach, and the other set of plants were used to measure high-affinity nitrate influx rates in a nutrient solution containing 0.2 mmol/L 15N-labeled nitrate. The results showed that exogenous ABA induced the expression of the TaNRT2.1, TaNRT2.2, TaNRT2.3, TaNAR2.1, and TaNAR2.2 genes in roots when nitrate was not present in the nutrient solution, but did not further enhance the induction of these genes by nitrate. Glutamine, which has been shown to inhibit the expression of NRT2 genes when nitrate is present in the growth media, did not inhibit this induction. When Gln was supplied to a nitrate-free nutrient solution, the expression of these five genes in roots was induced. These results imply that the inhibition by Gln of NRT2 expression occurs only when nitrate is present in the growth media. Although exogenous ABA and Gln induced HATS genes in the roots of wheat, they did not induce nitrate influx.