The mechanisms involved in regulating high-affinity ammonium (NH4+) uptake and the expression of the AtAMT1 gene encoding a putative high-affinity NH4+ transporter were investigated in the roots of Arabidopsis thaliana. Under conditions of steady-state nitrogen (N) supply, transcript levels of the AtAMT1 gene and Vmax values for high-affinity 13NH4+ influx were inversely correlated with levels of N provision. Following re-supply of NH4NO3 to N-starved plants, AtAMT1 mRNA levels and 13NH4+ influx declined rapidly but remained high when the conversion of NH4+ to glutamine (Gln) was blocked with methionine sulfoximine (MSX). This result demonstrates that end products of NH4+ assimilation, rather than NH4+ itself, are responsible for regulating AtAMT1 gene expression. Consistent with this hypothesis, AtAMT1 gene expression and NH4+ influx were suppressed by provision of Gln alone, or together with NH4NO3 plus MSX. Furthermore, AtAMT1 transcript levels and 13NH4+ influx were negatively correlated with root Gln concentrations, following re-supply of N to N-starved plants. In addition to this level of control, the data suggest that high cytoplasmic [NH4+] may inhibit NH4+ influx.