The glucagon receptor mediates the actions of glucagon on carbohydrate metabolism by the liver and on insulin release by the pancreatic beta -cell, which are key processes in the control of glucose homeostasis. The 5 ' -region of the mouse glucagon receptor gene has been recently cloned and two functional promoters were characterized. In the present study, we show that most of the glucagon receptor mRNA was transcribed from the distal promoter, in the mouse liver. In the distal promoter region, a GC-rich sequence with five putative binding sites for the Sp family of transcription factors was localized. To elucidate the role of these Sp1-binding sites in the mouse MIN6 beta -cell line, the expression of reporter gene constructs containing deletion or point mutation of each site was carried out. Selective mutation of the second Sp1-binding site decreased the activity of the distal promoter. Electrophoretic mobility shift assay with a DNA fragment spanning the three first Sp1 sites confirmed that the second site bound specifically MIN6 nuclear proteins, and supershift using specific Sp antibodies demonstrated that it interacted with Sp3 but not Sp1 transcription factors. These data illustrate that the basal expression of the mouse glucagon receptor gene, driven by the distal promoter, requires an Sp1-binding site that binds Sp3 proteins.