Abstract13N‐Iabelled experiments were carried out with barley and squash seedlings to analyse the effect of methionine sulphoximine (MSO), as an inhibitor of the ammonium assimilation, on NH4+fluxes in the roots at the level (150 μM NH4+) of the high affinity uptake system (HATS). MSO pretreatment in the root medium significantly accelerated NH4+influx and efflux in NH4+‐fed plants of both species. Concomitantly, the level of NH4+extracted from the root and shoot tissues was strongly elevated whereas the translocation of13N‐Iabelled organic substances (presumably amino products) into the shoots was markedly reduced. The onset of the increased NH4+influx did not occur immediately but was indicated about 1 h after the application of MSO to the nutrient solution. Also, MSO did not directly affect the uptake of13NO3−. The reason why MSO treatment leads to an acceleration of the NH4+influx against an increasing level in the cell tissue remains obscure. In squash, an intermediate of NO3−metabolism is thought to suppress NH4+uptake since NH4+influx was high in N‐free‐ and NH4+‐grown seedlings but was reduced about threefold in NO3−‐fed squash plants; the suppression was not elimina