Pancreatic beta-cell-type-specific transcription of the insulin gene is controlled by cis-acting sequence elements lying within its enhancer region. An essential element required for expression is the insulin control element (ICE). The activity of this element is regulated by both positive- and negative-acting transcription factors. In this study, we have identified the nucleotide sequences within the ICE that are required for repression in noninsulin producing cells. Our results indicate that the cis-acting sequences involved in negative control are distinct from those required in activating expression in beta cells.