Cell specific expression of the insulin gene is achieved through transcriptional mechanisms operating on 5' flanking DNA elements. In the enhancer of rat I insulin gene, two elements, the Nir and Far boxes, located at positions -104 and -233 respectively and containing the same octameric motif are essential for B cell specific transcription activity. Homologous sequences are present in the human insulin gene. While studying the binding of nuclear proteins from insulinoma cells to the -258/+241 region of the human insulin gene, we observed a previously undetected protein binding site in the intron I region between nucleotides +160 and +175. The binding activity was present in insulin producing cells such as RIN and HIT insulinoma cells but not in fibroblasts or insulin negative fibroblast x RIN hybrid cells. DNAse I footprinting and gel retardation/methylation interference experiments allowed us to define the core binding site of the intron binding factor as a GGGCCC hexamer. This factor is also capable to bind to a related sequence, contiguous to the Far-like element in rat and human insulin genes. The binding of the GGGCCC binding factor in this critical region of the insulin gene enhancer may participate in the regulation of insulin gene expression.