The chipmunk hibernation-specific protein HP-55 is a component of a 140-kDa complex whose levels are drastically decreased in the blood during hibernation. It is highly homologous to alpha(1)-antitrypsin (AT). In the chipmunk, several alpha(1)-AT-like genes in addition to HP-55 (or CM55-ML) are expressed in the liver and have distinct patterns of regulation during hibernation: in hibernating chipmunks, the level of CM55-ML gene expression is greatly reduced, that of the CM55-MS gene is slightly increased, and the expression of the CM55-MM gene is hardly affected. As a first step towards understanding the hibernation-associated gene regulation of these chipmunk alpha(1)-AT-like genes, we isolated genomic clones for the CM55-ML, CM55-MM, and CM55-MS genes, and analyzed their promoter activities. These alpha(1)-AT-like genes are composed of five exons, and show a similar gene structure to that of the human alpha(1)-AT gene, suggesting that they were generated by the duplication of an ancestral alpha(1)-AT gene. Transient transfection studies using HepG2 and COS-7 cells revealed that for all three alpha(1)-AT-like genes, approximately 150-bp 5' flanking sequences were sufficient for the liver-specific promoter activity, and that the binding of HNF-1 to the promoter region could transactivate transcription. In addition, analysis of the activity of chimeric promoters composed of CM55-ML and CM55-MS gene sequences indicated that the lack of a TATA box-like sequence in the CM55-MS gene is responsible for its weak promoter activity.