We have cloned and characterized the rat gene that encodes the p48 DNA-binding subunit of pancreas transcription factor 1 (Ptf1), a cell-specific basic region helix-loop-helix (bHLH) protein. The ptf1-p48 gene measures 1.8 kilobases in size and occurs as a single copy in the haploid genome. Run-on transcription assays suggest that this gene is subject to transcriptional control since no activity of its promoter is detected in nonproducing cells. The gene specifies two mRNAs that encode the same protein and originate from transcription initiation at alternative sites. Expression analysis of hybrid genes bearing deletions of the gene's 5'-flanking region fused to a reporter gene defines a promoter region within the gene-proximal 260 base pairs of DNA. The cis-acting elements that control promoter activity include binding sites for transcription factors Sp1 and alphaCbf, a 60-kDa CCAAT box-binding protein. The gene promoter, however, functions not only in exocrine pancreatic cells but also in cells of other origin. No cell-specific transcriptional control element was detected in as much as 10 kilobases of 5'-flanking region. We discuss models of how the cell-specific expression of the endogenous ptf1-p48 gene might be established during development of the animal.