The gastrin-releasing peptide (GRP) is a neuropeptide hormone and growth factor produced normally by neural and neuroendocrine cells, as well as by human small-cell lung cancer (SCLC) tumors and derived cell lines. This study compares the structure of the human prepro-GRP gene in four SCLC cell lines that express variable levels of steady-state GRP mRNA. The regulation of GRP gene expression appears to be at the level of primary transcription based on nuclear run on studies. In the two SCLC cell lines expressing GRP we find a single transcription start site for GRP mRNA, and near this site we find four DNase I hypersensitive sites. These hypersensitive sites are absent in the two cell lines that do not express GRP. The presence of DNase hypersensitive sites in the promoter region of the GRP gene is the structural feature that best correlates with transcriptional activation. These four DNase hypersensitive sites are candidates for cis acting regulatory regions, which may be important in determining the level of transcription of the human prepro GRP gene.