We cloned a cDNA for Drosophila mitochondrial transcription factor A (D-mtTFA) and characterized the recombinant protein. In Drosophila Kc cells, D-mtTFA was localized in the mitochondria, but not in the nucleus. By repetitive precipitation with His-tag and PCR amplification, the consensus nucleotide sequence for D-mtTFA-binding was determined to be 5 ' -TTATC/G. The binding sequence was found to be clustered in the A + T region of mitochondrial DNA which is suggested to be a replication origin and promoter region for light strand and heavy strand. We found a DNA replication-related element (DRE)-like sequence located upstream of the transcription initiation site of the D-mtTFA gene and obtained results indicating that DRE-binding factor (DREF) can bind to the DRE-like sequence of the D-mtTFA gene. The data suggest that transcription of the D-mtTFA gene is under control of the DRE/DREF regulatory system. Based on these results, the functions of D-mtTFA were discussed in relation to mitochondrial biogenesis of Drosophila melanogaster.