We previously reported cloning of the Drosophila mitochondrial transcription factor A (D-mtTFA) gene, and characterization of a recombinant protein. In this report, the expression and distribution patterns of D-mtTFA during development are described. D-mtTFA mRNA and its protein product were found to be abundant in cells of tissues undergoing both proliferation and polytenization. Furthermore, a DRE-like sequence present in the D-mtTFA gene showed promotor activity in Drosophila Kc cells. In addition, D-mtTFA was detected in brain throughout the developmental process, as well as in non-proliferating tissues, such as flight muscle and cardia, and was also found in spermatids of imagos. D-mtTFA possesses a nuclear-targeting sequence, and is present in the nucleus at the syncytial stage, where bundles of 64 spermatids are present during spermatogenesis. The results suggest that D-mtTFA not only contributes to the mitochondrial DNA transcription and replication system, but also has a role in cell proliferation and development, including spermatogenesis.