The B element of the elastase I transcriptional enhancer is active in both exocrine and endocrine cells of the pancreas. Cell transfection experiments revealed that in an acinar cell line the active sequence of the element is more extensive than in an endocrine cell line. Electrophoretic mobility shift assays identified three major complexes (designated C, M, and L) from acinar cell nuclear extracts bound to the element. The C complex appears to be responsible for the activity of the element in acinar cells because its binding site, determined by methylation interference and mobility shift competition experiments, matches the critical sequence identified by cell transfection analysis of mutated B elements. The DNA sequence requirements for formation of the C complex is the sum of those for the M and L complexes. Methylation interference experiments indicated that the sensitivity of the C complex to guanine methylation also was the sum of that of the M and L complexes. Diagonal electrophoretic mobility shift assays confirmed that L is a component of complex C. However, the M complex, which we identified as GATA-4, is not part of the C complex, because the C complex was neither competed by GATA-binding sites nor super-shifted by anti-GATA-4 antiserum. Both the C and L complexes are specific to the pancreatic acinar cell line.