The human asparagine synthetase (AS) gene is transcriptionally regulated by amino acid deprivation (amino acid response, AAR) and the endoplasmic reticulum stress response (ERSR), also known as the unfolded protein response pathway. The results reported here document the novel observation that induction of the AS gene by the AAR and ERSR pathways occurs via the same set of genomic elements. Data supporting this conclusion include transient transfection of AS promoter/reporter gene constructs that illustrate that the transcriptional control elements used by both pathways are contained with nucleotides -111 to -34 of the AS promoter. In vivo footprinting analysis of this region identified six specific protein-binding sites. Within two of these sites, altered footprinting was observed following amino acid or glucose deprivation, but the patterns were identical for both the AAR and the ERSR pathway. Site-directed mutation of individual nucleotides within these two binding sites confirmed their importance for regulated transcription, and none of the mutations resulted in loss of response of only one pathway. Neither of these two sites corresponds to a recently identified ERSR cis-element, nor do they contain consensus sequences for known transcription factors. Collectively, the data document that there are at least two independent transcriptional mechanisms for gene activation by the ERSR pathway, one of which terminates at the same genomic elements used by the AAR pathway.