Two separate domains within the 134-base-pair rat elastase I enhancer and a third domain at the enhancer-promoter boundary are required for selective expression in pancreatic acinar cells. The domains were detected by a series of 10-base-pair substitution mutations across the elastase I gene regulatory region from positions -200 to -61. The effect of each mutant on the pancreas-specific expression of a linked chloramphenicol acetyltransferase gene was assayed by transfection into pancreatic 266-6 acinar cells and control NIH/3T3 cells. The two enhancer domains are nonredundant, because mutations in either eliminated (greater than 100-fold reduction) expression in 266-6 cells. DNase I protection studies of the elastase I enhancer-promoter region with partially purified nuclear extracts from pancreatic tissue and 266-6 cells revealed nine discrete protected regions (footprints) on both DNA strands. One of three footprints that lie within the two functional domains of the enhancer contained a sequence, conserved among several pancreas-specific genes, which when mutated decreased linked chloramphenicol acetyltransferase expression up to 170-fold in 266-6 cells. This footprint may represent a binding site for one or more pancreas-specific regulatory proteins.