Substantial concentrations of NH4+ are found in the apoplast of the leaves of Brassica napus. Physiological studies on isolated mesophyll protoplasts with 15NH4+ revealed the presence of a high-affinity ammonium transporter that shared physiological similarity to the high-affinity NH4+ transporters in Arabidopsis thaliana (AtAMT1;3). PCR techniques were used to isolate a full-length clone of a B. napus homologue of AMT1 from shoot mRNA which showed 97% similarity to AtAMT1;3. The full-length cDNA when cloned into the yeast expression vector pFL61 was able to complement a yeast mutant unable to grow on media with NH4+ as the sole nitrogen source. Regulatory studies with detached leaves revealed a stimulation of both NH4+ uptake and expression of mRNA when the leaves were supplied with increasing concentrations of NH4+. Withdrawal of NH4+ supply for up to 96 h had little effect on mRNA expression or NH4+ uptake; however, plants grown continuously at high NH4+ levels exhibited decreased mRNA expression. BnAMT1;2 mRNA expression was highest when NH4+ was supplied directly to the leaf and lowest when either glutamine or glutamate was supplied to the leaves, which directly paralleled chloroplastic glutamine synthetase (GS2) activity in the same leaves. These results provide tentative evidence that BnAMT1;2 may be regulated by similar mechanisms to GS2 in leaves.