The physiological effects of glucose-dependent insulinotropic polypeptide (GIP) are mediated through specific receptors expressed on target cells. Because aberrant GIP receptor (GIPR) expression has been implicated in abnormal GIP responses associated with type II diabetes mellitus and food-induced Cushing[[rad]]s syndrome, we sought to identify factors that regulate the GIPR. We previously demonstrated that sequences between -1 and -100 of the GIPR gene were sufficient to direct transcription in a rat insulinoma cell line (RIN38). In the present study, we compared the 5'-flanking regions of the rat and human GIPR gene and demonstrated 88% identity within the first 92 base pairs. Subsequent serial deletion analyses showed that the region between -85 and -40 is essential for maximal promoter activity. Within this region, we identified three putative Sp1 binding motifs, located at positions -77, -60 and -50, that can specifically bind both Sp1 and Sp3. Whereas mutation of the Sp1 sites at -50 and -60 led to 36% and 40% reduction in promoter activity, respectively, mutation of the Sp1 motif at -70 did not affect promoter activity. Co-transfection of S2 Schneider cells with GIPR-luciferase chimeric constructs and either Sp1 or Sp3 expression vectors indicated that both Sp1 and the long-form of Sp3 activate transcription through binding to the Sp1 sites located between -100 and -40. Lastly, chromatin immunoprecipitation analyses revealed that both Sp1 and Sp3 bind to the GIPR promoter region in RIN38 cells. These results indicate that cell-specific expression of the GIPR is associated with the binding of the transcription factors Sp1 and Sp3 to the GIPR promoter.