The hibernation-specific HP-27 gene is expressed specifically in the liver of the chipmunk, a hibernating species of the squirrel family, and exists as a pseudogene in the tree squirrel, a nonhibernating species. In the promoter region, the chipmunk gene has a potential HNF-1 binding site, and the tree squirrel gene has two base substitutions in the corresponding sequence. In this paper, we investigated the role of HNF-1 in the HP-27 gene promoter activity. Gel retardation assays with in vitro-translated HNF-1 and super-shift assays using HepG2 nuclear extracts and an anti-HNF-1 antibody revealed that HNF-1 bound to the chipmunk gene sequence. HNF-1 also bound to the tree squirrel sequence, but with much lower affinity. In HepG2 cells, HNF-1 activated transcription from the chipmunk HP-27 gene, but not from the tree squirrel gene. In addition, the tree squirrel-type base substitutions in the HNF-1 binding site greatly reduced the promoter activity of the chipmunk HP-27 gene. These results indicate that HNF-1 is required for the promoter activity of the chipmunk HP-27 gene, and that the base substitutions in the HNF-1 binding site are involved in the lack of HP-27 gene expression in the tree squirrel.