The human pancreatic secretory trypsin inhibitor (PSTI) genes introduced into mice are specifically expressed in pancreas. The 1.0 kilobase pairs of PSTI 5'-flanking sequence directed preferential expression of a linked reporter chloramphenicol acetyltransferase, which was active in a PSTI-expressing pancreatic cell line (AR42j) but not in a PSTI-nonexpressing fibroblast cell line (XC). Two positively acting elements were found, Region I (-161/-116) and Region II (-103/-74), as defined by transfection and binding assays with AR42j cells. Region II is sufficient for the pancreas-specific expression, but the presence of both Regions I and II is needed for the maximum activity. Sequence studies also revealed that these two elements differ from the previously identified recognition sequence for pancreas transcription factor 1 (PTF1). When the same set of experiments was done with XC cells, one negatively acting element was identified, Region IV (-154/-137). Interestingly, Regions I and IV share a core sequence (-149/-139), CAATCAATAAC. These results suggest that this novel element regulates the human PSTI gene expression positively in pancreatic cells but negatively in nonpancreatic cells.