Sequence analysis of the 5′flanking region of the β-cellspecific transcription unit of the rat glucokinase gene (rβGK) revealed the presence of sequence motifs very similar to the IEB-(Far)-box and a CT-motif which play a crucial role in transcriptional control of insulin genes. 5′deletional analysis of the rβGK proximal promoter element (localized between nucleotides -278 and -49) as well as site directed mutagenesis showed that both motifs are mutationally sensitive and contribute to transcriptional control in HIT M2.2.2 cells. The combination of the IEB-(Far)-like motif with the CT-box was unable to form a “mini-enhancer” similar to the Far-FLAT-element of the rat insulin I gene promoter but rather functions as a β-cell specific control element in rβGK expression. Electrophoretic mobility shift assays (EMSAs) and competition studies using oligonucleotides containing CT-motifs of rat insulin genes promoters, human insulin gene promoter, and rat amylin gene promoter showed similar binding patterns with nuclear extracts isolated from insulin-producing cell lines. These studies indicate that CT-motifs of rat glucokinase, insulin, and amylin gene promoters may bind similar - probably identical - nuclear factor(s) and may play a central role in the coordinated expression of these genes in insulin-producing cells.