Hepatocarcinoma Intestine Pancreas (HIP) gene, also called Pancreatitis Associated Protein 1 (PAP1) or Reg IIIalpha, is activated in most human hepatocellular carcinomas (HCC) but not in normal liver, which suggests that HIP regulatory sequence could be used as efficient liver tumor-specific promoters to express a therapeutic polynucleotide in liver cancer. Sodium Iodide Symporter (NIS), which has recognized therapeutic and reporter gene properties, is appropriate to evaluate the transcriptional strength and specificity of HIP promoter in HCC. For this purpose, we constructed a recombinant rat HIP-NIS adenovirus vector (AdrHIP-NIS), and evaluated its performance as a mediator of selective radioiodide uptake in tumor hepatocytes. Western blot, immunofluorescence, and iodide uptake assays were performed in AdrHIP-NIS-infected primary hepatocytes and transformed hepatic and non-hepatic cells. Nuclear imaging, tissue counting and immunohistochemistry were performed in normal and HCC-bearing Wistar rats infected with AdrHIP-NIS in tumor nodules or via the hepatic artery. In AdrHIP-NIS-infected transformed hepatic cells, a functional NIS was strongly expressed, as in cells infected with a cytomegalovirus-NIS vector. No NIS expression was found in AdrHIP-NIS-infected normal hepatocytes or transformed non-hepatic cells. In rats bearing multinodular HCC, AdrHIP-NIS triggered functional NIS expression which was preferential in tumor hepatocytes. The administration of 18mCi 131I resulted in the destruction of AdrHIP-NIS-injected nodules. This study has identified the rHIP regulatory sequence as a potent liver tumor-specific promoter for the transfer of therapeutic genes, and AdrHIP-NIS-mediated 131I therapy as a valuable option for the treatment of multinodular HCC.