The 100-base pair ELA1 transcriptional enhancer drives high level transcription to pancreatic acinar cells of transgenic mice and in transfected pancreatic acinar cells in culture. The A element within the enhancer is the sole positively acting element for acinar specificity. We show that the acinar cell-specific bHLH protein PTF1-P48 and the common bHLH cofactor HEB are part of the PTF1 complex that binds the A element and mediates its activity. Acinar-like activity of the enhancer can be reconstituted in HeLa cells by the introduction of P48, HEB, and the PDX1-containing trimeric homeodomain complex that binds the second pancreatic element of the enhancer. The 5' region of the mouse Ptf1-p48 gene from -12.5 to +0.2 kilobase pairs contains the regulatory information to direct expression in transgenic mice to the pancreas and other organs of the gut that express the endogenous Ptf1-p48 gene. The 5'-flanking sequence contains two activating regions, one of which is specific for acinar cells, and a repressing domain active in non-pancreatic cells. Comparison of the 5'-gene flanking regions of the mouse, rat, and human genes identified conserved sequence blocks containing binding sites for known gut transcription factors within the acinar cell-specific control region.