Background: Suicide gene therapy has been shown to be an effective means of destroying pancreatic cancer cells, but cell specific delivery of the gene is required to limit host toxicity. The objective of this study is to determine whether the rat insulin promoter (RIP) will permit cell specific gene delivery and subsequent cell death in human pancreatic cancer cells. Methods: The RIP DNA was amplified using PCR method and the purified fragment was inserted into pCR-Blunt II-TOPO plasmid at SpeI site which contains the coding sequence of yeast cytosine deaminase (CD). Transfection assays were carried out using both RIP-lacZ and RIP-CD DNA constructs into two human pancreatic cancer cell lines, Panc-1 and MIA PaCa-2. Reporter assays using X-gal staining were performed and the in vitro cytotoxicity was examined in RIP-CD transfected cells which was treated by 5-FC (5-flucytosine) for 5 days. The expression levels of CD protein in the transfected cells were determined 2 days post transfection by western blot analysis. The expression levels of insulin promoter factor (IPF-1/PDX-1) in these human pancreatic cell lines as well as freshly isolated human specimens were determined using western blot analysis. Results: Western blot analysis demonstrated that significantly increased levels of PDX-1 were found in Panc-1 but not Mia PaCa-2 cells. X-gal staining showed that only PANC-1 cells, not MIA PaCa-2 cells, were positive for RIP-lacZ expression indicating that the insulin promoter directed reporter gene expression in Panc-1 cells, but not in MIA PaCa-2. RIP-CD transfected and 5-FC treated Panc-1 cells had significantly increased cell death compared with that of MiaPaca-2 cells, suggesting RIP directed suicide gene expression occurred in Panc-1 but not Mia PaCa-2 cells. Western blot analysis demonstrated that only Panc-1 cells, not MIA PaCa-2 cells, were able to express the CD protein. Furthermore, six freshly isolated human pancreatic cancer specimens and two pancreatic cancer liver metastases also had significantly increased levels of PDX-1. Conclusion: The rat insulin promoter is activated in selected human pancreatic cancer cells that express PDX-1. Significantly increased levels of PDX-1 have been found in 8/8 human pancreatic cancer specimens. Pancreatic cancer-specific cytotoxicity can be achieved using RIP-CD and 5-FC treatment in vitro. These results suggest that RIP could be used for cell specific gene therapy to target human pancreatic tumors.