Phosphoenolpyruvate carboxykinase (PEPCK) is one of the key regulatory enzymes in gluconeogenesis. In human liver, PEPCK is about equally distributed in both cytosol (PEPCK-1) and mitochondria (PEPCK-2). The human pepck2 gene and cDNA have been reported, but the cloning of the promoter region of the pepck2 gene has not been elucidated yet. We isolated and characterized human genomic P1-artificial chromosome (PAC) clones carrying the human pepck2 gene promoter. The oligocapping method revealed that the transcriptional start point (tsp) of the human pepck2 gene is located at 97 bp upstream of the first adenine residue of the translation start site. We also determined the nucleotide sequence to 1819 bp upstream of tsp. Sequence analysis of this region revealed that it contained several potential regulatory elements, including five GC boxes and three CCAAT boxes. Reporter analysis using transient transfection with firefly luciferase synthetic gene indicated 5' flanking region up to 822 bp, and 317 bp upstream of tsp had transcriptional activity. These results suggest that these regions of the human pepck2 gene play an important role for its expression.