A soybean transcription factor, Glycine max bHLH membrane 1 (GmbHLHm1), was previously shown to activate an uncharacterized gene in yeast, later named Saccharomyces cerevisiae ammonium facilitator 1 (ScAMF1). ScAMF1 homologs are common in plants including Arabidopsis thaliana. The functions of AMFs in plants are unknown. Previous experiments in yeast and Xenopus laevis oocytes have shown that AMFs from yeast and soybean are able to transport ammonium. To determine the function of AMFs in Arabidopsis, transcriptional analysis and reverse genetic approaches using T-DNA knockout lines were employed. Quantitative PCR experiments showed that three AtAMF genes have higher expression in older leaves than in young leaves and root tissue, with AtAMF2 (At5g64500) having the highest expression followed by AtAMF3 (At5g65687) and AtAMF1 (At2g22730), respectively. Additionally, all AtAMFs were diurnally regulated and responded to nitrogen starvation and resupply. Growth analysis of homozygous single knockout mutants (amf1, amf2 and amf3) grown hydroponically in nutrient solution with 1 mM NH4NO3 for 7 weeks showed a decrease in root dry weight but not shoot dry weight compared to wild type plants. However the photosynthetic rate, stomatal conductance and respiration rate were not significantly different between lines. Tissue localization using GUS driven by a native promoter revealed that AtAMF1 is localized in guard cells and phloem, AtAMF2 in the root tip and AtAMF3 in phloem tissue. An interesting phenotype appeared in a double knockout mutant amf3/amf2, which showed reduced root length when plants were grown on agar plates without the addition of sucrose. More studies need to be done to empirically characterize the function of AMF including electrophysiology, yeast complementation, and metabolomic studies.