The human asparagine synthetase (AS) gene responds to depletion of mammalian cells for either amino acids or carbohydrates. Five specific cis-elements have been implicated: three GC boxes (GC-I, GC-II and GC-III) and two nutrient-sensing response elements (NSRE-1, -2). This study shows that all three GC boxes are required to maintain basal transcription and to obtain maximal induction of the AS gene by amino acid limitation. However, there is not complete redundancy among the three GC boxes, and there is a hierarchy of importance with regard to transcription (GC-III > GC-II > GC-I). In vitro, two GC boxes formed protein-DNA complexes (GC-II and GC-III) with Sp1 and Sp3. Although transcription of the AS gene is elevated by nutrient limitation, the absolute amount of these protein-DNA complexes and the total pools of Sp1 and Sp3 did not increase. A small, but detectable portion of Sp1 was modified by phosphorylation following amino acid deprivation. In vivo, expression of Sp1 and Sp3 in Drosophila SL2 cells increased AS promoter activity. Sp1 expression increased basal transcription but did not cause a further increase when SL2 cells were amino acid-deprived. Sp3 expression enhanced both the basal and the starvation-induced transcription.