The high-affinity nitrate transporter of green plants is composed of two polypeptides, NRT2.1 and NAR2.1, while in fungi it appears that nitrate influx is mediated by NRT2 alone. Another difference between plants and fungi is that the central (cytoplasmic) loop of the 12 membrane spanning regions of NRT is quite large in fungi, consisting of 91 amino acid residues, compared with the relatively short (21 amino acid residues) plant NRT2.1. Here we examine potential amino acid residues involved in the plant NRT2.1:NAR2.1 association by mutation of conserved amino acids in Arabidopsis thaliana AtNRT2.1. Only the replacement of leucine 85 by glutamine disrupted the association between AtNRT2.1 and AtNAR2.1, as examined using the yeast two-hybrid system. Further, to investigate the nitrate-transporting function of AtNRT2.1 in a context free of other members of the NRT2 family, we expressed AtNRT2.1 in Aspergillus nidulans. In the fungal context the plant NRT alone was capable of restoring nitrate transport to a nitrate transport defective mutant, but only when the AtNRT2.1 central loop was replaced by its fungal counterpart.