Real-time polymerase chain reaction analysis was used to compare the effect of NO − 3 on the activities of nitrate reductase (NR) and glutamine synthetase (GS), and the transcript levels of two NR genes, OsNia1 and OsNia2, two cytosolic GS1 genes, OsGln1;1 and OsGln1;2, and one plastid GS2 gene OsGln2, in two rice ( Oryza sativa L.) cultivars Nanguang (NG) and Yunjing (YJ). Both cultivars achieved greater biomass and higher total N concentration when grown in a mixed N supply than in sole NH + 4 nutrition. Supply of NO − 3 increased NR activity in both leaves and roots. Expression of both NR genes was also substantially enhanced and transcript levels of OsNia2 were significantly higher than those of OsNia1. NO − 3 also caused an increase in GS activity, but had a complex effect on the expression of the three GS genes. In roots, the OsGln1;1 transcript increased, but OsGln1;2 decreased. In leaves, NO − 3 had no effect on the GS1 expression, but the transcript for OsGln2 increased both in the leaves and roots of rice with a mixed supply of N. These results suggested that the increase in GS activity might be a result of the complicated regulation of the various GS genes. In addition, the NO − 3-induced increase of biomass, NR activity, GS activity, and the transcript levels of NR and GS genes were proportionally higher in NG than in YJ, indicating a stronger response of NG to NO − 3 nutrition than YJ.