The pancreatic β cell makes several unique gene products, including insulin, islet amyloid polypeptide (LAPP), and β-cell-specific glucokinase (βGK). The functions of isolated portions of the insulin, IAPP, and βGK promoters were studied by using transient expression and DNA binding assays. A short portion (—247 to —197 bp) of the rat insulin I gene, the FF minienhancer, contains three interacting transcriptional regulatory elements. The FF minienhancer binds at least two nuclear complexes with limited tissue distribution. Sequences similar to that of the FF minienhancer are present in the 5′ flanking DNA of the human LAPP and rat βGK genes and also the rat insulin II and mouse insulin I and II genes. Similar minienhancer constructs from the insulin and IAPP genes function as cell-specific transcriptional regulatory elements and compete for binding of the same nuclear factors, while the βGK construct competes for protein binding but functions poorly as a minienhancer. These observations suggest that the patterns of expression of the β-cell-specific genes result in part from sharing the same transcriptional regulators.