The developmental pattern of expression of the genes encoding the catalytic (alpha) and accessory (beta) subunits of mitochondrial DNA polymerase (pol gamma) has been examined in Drosophila melanogaster. The steady-state level of pol gamma-beta mRNA increases during the first hours of development, reaching its maximum value at the start of mtDNA replication in Drosophila embryos. In contrast, the steady-state level of pol gamma-alpha mRNA decreases as development proceeds and is low in stages of active mtDNA replication. This difference in mRNA abundance results at least in part from differences in the rates of mRNA synthesis. The pol gamma genes are located in a compact cluster of five genes that contains three promoter regions (P1-P3). The P1 region directs divergent transcription of the pol gamma-beta gene and the adjacent rpII33 gene. P1 contains a DNA replication-related element (DRE) that is essential for pol gamma-beta promoter activity, but not for rpII33 promoter activity in Schneider's cells. A second divergent promoter region (P2) controls the expression of the orc5 and sop2 genes. The P2 region contains two DREs that are essential for orc5 promoter activity, but not for sop2 promoter activity. The expression of the pol gamma-alpha gene is directed by P3, a weak promoter that does not contain DREs. Electrophoretic mobility shift experiments demonstrate that the DRE-binding factor (DREF) regulatory protein binds to the DREs in P1 and P2. DREF regulates the expression of several genes encoding key factors involved in nuclear DNA replication. Its role in controlling the expression of the pol gamma-beta and orc5 genes establishes a common regulatory mechanism linking nuclear and mitochondrial DNA replication. Overall, our results suggest that the accessory subunit of mtDNA polymerase plays an important role in the control of mtDNA replication in Drosophila.