Keratin 7 is expressed in simple epithelia but is expressed at low or undetectable levels in gastrointestinal epithelial cells. In the pancreas, it is present in ductal but not in acinar cells. K7 mRNA is overexpressed in pancreatic cancers. Here we use luciferase reporter assays to analyze the tissue-specific regulatory elements of murine keratin 7 (Krt7) promoter in vitro and in vivo. All elements required for appropriate cell and tissue specificity in reporter assays are present within the Krt7 -234 bp sequence. This fragment appears more selective to pancreatic ductal cells than the Krt19 promoter. GC-rich sequences corresponding to putative Sp1, AP-2 binding sites are essential for in vitro activity. Krt7-LacZ transgenic mice were generated to analyze in vivo activity. Sequences located 1.5 or 0.25 kb upstream of the transcription initiation site drive reporter expression to ductal, but not acinar, cells in transgenic mice. LacZ mRNA was detected in the pancreas as well as in additional epithelial tissues--such as the intestine and the lung--using both promoter constructs. An AdK7Luc adenovirus was generated to assess targeting selectivity in vivo by intravenous injection to immunocompetent mice and in a xenograft model of pancreatic cancer. The -0.25 kb region showed pancreatic selectivity, high activity in pancreatic cancers, and sustained transgene expression in xenografts. In conclusion, the krt7 promoter is useful to target pancreatic ductal adenocarcinoma cells in vitro and in vivo.