Exocrine pancreas (XP) enhancers, which contain a conserved core sequence, are active only in XP cells. A core enhancer-binding activity also appears to be restricted to XP nuclei. Here we describe the properties of a factor, purified approximately 100,000-fold from pancreas nuclei, which displays core enhancer-binding activity. It is not identical to previously characterized factors and is termed exocrine pancreas transcription factor 1 (XPF-1). In the highly purified preparation, only a single major protein of 60 kDa was detected by silver staining on sodium dodecyl sulfate-gels and by UV cross-linking. XPF-1 binds to the core enhancer of all tested XP genes and not to a mutant sequence which is inactive in vivo. High-affinity binding sites are bipartite. The results of competition binding and UV-cross-linking assays suggest that XPF-1 interacts with both motifs. XPF-1 selectively stimulates transcription of core enhancer templates in an in vitro transcription system. We hypothesize that XPF-1 plays a role in activation of the transcription of XP-specific genes.The differentiated phenotype presumably reflects the selective expression of a set of genes whose products carry out the specialized functions of a given cell. While various steps in gene expression may be subject to regulation, the initiation of transcription appears to be the major level of control for many cell-specific genes (6, 31). Transcriptional regulators bind to specific DNA sequence elements and influence the rate of initiation (34,48). Understanding the molecular basis of this regulatory process requires identification of the components and characterization of their interactions in the functional transcription complex.As a paradigm of transcriptional control mechanisms in the differentiated phenotype, we have studied gene expression in the acinar cells of the exocrine pancreas (XP). These cells synthesize copious quantities of the digestive enzymes (50), and the genes encoding these products are expressed at high levels in a tissue-specific fashion. To a large degree, expression is mediated at the level of transcription initiation (11,29). cis-acting DNA sequences which confer XP-specific transcription initially were identified in 5' flanking sequences of the chymotrypsin B gene by using transient transfection of an XP cell line (59) and of the elastase I gene by using transgenic mice (39,40,55). In subsequent gene transfer experiments, it was shown that transcriptional control of these and the amylase 2A and trypsin I genes is mediated by XP-specific enhancers (1,13,42).Sequences required for XP enhancer function were defined by deletion and substitution analysis (1,4,13,17,23,26,32,42,56). These studies demonstrated the importance of a 20-nucleotide conserved sequence (the pancreatic consensus) present in each of the identified XP-specific enhancers and in the 5' flanking region of other XP genes (1,5,10,52,54). In transient transfection experiments, this conserved sequence is required for amylase 2A, chymotrypsin B, and elastase I enhance...