Adenine nucleotide translocase (ANT) is a crucial component in the maintenance of cellular energy homeostasis, as well as in the formation of the mitochondrial permeability transition pores. However, the molecular mechanisms regulating the expression of the ANT gene are poorly understood. In this study, we have identified three DNA replication-related elements (DRE; 5'-TATCGATA) in the 5'-flanking region of the Drosophila ANT (dANT) gene. Gel-mobility shift analyses revealed that all three of the DREs were recognized by the DRE-binding factor (DREF). The site-directed mutagenesis of these DRE sites induces a considerable reduction in the activity of the dANT gene promoter in vitro. Analyses with transgenic flies harboring a dANT-lacZ fusion gene bearing the wild-type or mutant DRE sites showed that the DRE sites were required for the expression of dANT in vivo. We determined that the over-expression or knockdown of DREF exerts a regulatory effect on the activity of the dANT promoter. In addition, we observed the collapse of mitochondrial membrane potential in the eye imaginal discs in which DREF was over-expressed. These results show that DRE/DREF is a crucial regulator of dANT gene expression, and also suggest the possibility that cross-talk may occur between the DRE/DREF system and mitochondrial functioning.