In plants, the amino acid asparagine serves as an important nitrogen transport compound whose levels are dramatically regulated by light in many plant species, includingArabidopsis thaliana. To elucidate the mechanisms regulating the flux of assimilated nitrogen into asparagine, we examined the regulation of the gene family for asparagine synthetase in Arabidopsis. In addition to the previously identifiedASN1gene, we identified a novel class of asparagine synthetase genes in Arabidopsis (ASN2andASN3) by functional complementation of a yeast asparagine auxotroph. The proteins encoded by theASN2/3cDNAs contain a Pur-F type glutamine-binding triad suggesting that they, likeASN1, encode glutamine-dependent asparagine synthetase isoenzymes. However, the ASN2/3 isoenyzmes form a novel dendritic group with monocotASgenes which is distinct from all other dicotASgenes including ArabidopsisASN1. In addition to these distinctions in sequence, theASN1andASN2genes are reciprocally regulated by light and metabolites. Time-course experiments reveal that light induces levels ofASN2mRNA while it represses levels ofASN1mRNA in a kinetically reciprocal fashion. Moreover, the levels ofASN2andASN1mRNA are also reciprocally regulated by carbon and nitrogen metabolites. The distinct regulation ofASN1andASN2genes combined with their distinct encoded isoenzymes suggest that they may play different roles in nitrogen metabolism, as discussed in this paper.