Production of insulin is stringently restricted to the beta cells of the endocrine pancreas. The major mechanism directing this specificity operates at the level of transcription. Previous studies have demonstrated that the tissue specificity in the insulin gene expression results from the cell-specific activity of its 5'-flanking enhancer/promoter, which limits the expression of a linked gene to the beta cell in tissue culture and transgenic mouse. Deletion, mutation, DNase I foot-printing, and gel-shift analyses revealed the presence of multiple cis-acting motifs. A set of positive and, possibly, negative trans-acting factors that are found in insulin-producing and noninsulin producing cells bind to these cis-acting elements and thus regulate transcription. Included among those are IEB (E-box)-binding IEF-1, a nuclear protein complex which consists of two basic helix-loop-helix proteins, and CT-box (TAAT box)-binding IPF1/STF-1, a homeodomain-containing transcription factor.