The effect of NH4+/NO3− availability on nitrate reductase (NR) activity in Phragmites australis and Glyceria maxima was studied in sand and water cultures with the goal to characterise the relationship between NR activity and NO3− availability in the rhizosphere and to describe the extent to which NH4+ suppresses the utilization of NO3− in wetland plants.The NR activity data showed that both wetland helophytes are able to utilize NO3−. This finding was further supported by sufficient growth observed under the strict NO3− nutrition. The distribution of NR activity within plant tissues differed between species. Phragmites was proved to be preferential leaf NO3− reducer with high NR activity in leaves (NRmax > 6.5 μmol NO2− g dry wt−1 h−1) under all N treatments, and therefore Phragmites seems to be good indicator of NO3− availability in flooded sediment. In the case of Glyceria the contribution of roots to plant NO3− reduction was higher, especially in sand culture. Glyceria also tended to accumulate NO3− in non-reduced form, showing generally lower leaf NR activity levels. Thus, the NR activity does not necessarily correspond with plant ability to take up NO3− and grow under NO3−-N source. Moreover, the species differed significantly in the content of compounds interfering with NR activity estimation. Glyceria, but not Phragmites, contained cyanogenic glycosides releasing cyanide, the potent NR inhibitor. It clearly shows that the use of NR activity as a marker of NO3− utilization in individual plant species is impossible without the precise method optimisation.