The ␤ cell-specific glucose-sensitive factor (GSF), which binds the A3 motif of the rat I and human insulin promoters, is modulated by extracellular glucose. A single mutation in the GSF binding site of the human insulin promoter abolishes the stimulation by high glucose only in normal islets, supporting the suggested physiological role of GSF in the glucose-regulated expression of the insulin gene. GSF binding activity was observed in all insulin-producing cells. We have therefore purified this activity from the rat insulinoma RIN and found that a single polypeptide of 45 kDa was responsible for DNA binding. Its amino acid sequence, determined by microsequencing, provided direct evidence that GSF corresponds to insulin promoter factor 1 (IPF-1; also known as PDX-1) and that, in addition to its essential roles in development and differentiation of pancreatic islets and in ␤ cell-specific gene expression, it functions as mediator of the glucose effect on insulin gene transcription in differentiated ␤ cells. The human cDNA coding for GSF͞IPF-1 has been cloned, its cell and tissue distribution is described. Its expression in the glucagon-producing cell line ␣TC1 transactivates the wild-type human insulin promoter more efficiently than the mutated construct. It is demonstrated that high levels of ectopic GSF͞IPF-1 inhibit the expression of the human insulin gene in normal islets, but not in transformed ␤TC1 cells. These results suggest the existence of a control mechanism, such as requirement for a coactivator of GSF͞ IPF-1, which may be present in limiting amounts in normal as opposed to transformed ␤ cells.The insulin gene contains several cis-acting regulatory elements located within its 5Ј-flanking region that are recognized by trans-acting factors, some found ubiquitously, others more restricted to the ␤ cell. These interactions determine the temporal expression of the gene and its inducibility by physiological stimuli (for review see refs. 1 and 2). Important transcriptional regulatory elements have been described in the promoter regions of various insulin genes, such as the E and A boxes (3). The motifs E1 and E2, with the consensus CANNTG, were found to be implicated in tissue-specific expression of insulin by transfection studies in insulinproducing cells. These bind transcription factors of the helixloop-helix family (4, 5). However, combinations of E1 and E2 fail to confer tissue specificity in transgenic mice (6). Additional important regulatory elements containing AϩT-rich sequences are the A boxes (A1-A5; ref.3). The proximal A1 box in the rat insulin I promoter (around Ϫ80) binds a protein selectively expressed in insulin-producing cell lines, insulin promoter factor 1 (IPF-1), which is a homeodomaincontaining transactivator of the insulin gene (7,8). By gene disruption in mice, it was shown that IPF-1 is of crucial importance for normal development of the pancreas (9). The distal A3 and A4 boxes bind several homeodomain-containing proteins, such as isl-1 (10), cdx-3 and lmx-1 (11), HNF1 ␣ (12...