A factor found in pancreatic exocrine cell lines and pancreatic nuclei binds selectively to the alpha-amylase 2A transcriptional enhancer. Pancreatic exocrine cell extracts protect asymmetrically an unusually large, 35 base pair region from DNase I digestion in vitro, suggesting the involvement of a multimeric DNA binding complex. We show that this region of the enhancer contains a major affinity recognition sequence for the HeLa transcription factor AP4. A 4 base pair mutation in the enhancer sequence shown previously to abolish activity in vivo [Boulet, A. M., Erwin, C. R., & Rutter, W. J. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3599-3603] abolishes AP4 binding in vitro and weakens but does not eliminate the binding of adjacent enhancer factors. Further, sequences similar to the AP4 binding site are found within a consensus sequence of most pancreatic exocrine genes (Boulet et al., 1986). We have identified three AP4 binding sites in the pancreatic elastase gene: one occurs in the consensus sequence of the enhancer. Thus, protein(s) with the binding selectivity of AP4 may play a role in the expression of the pancreatic exocrine gene family.