A full-length cDNA of the catalytic subunit of mitochondrial DNA polymerase from Drosophila embryos has been obtained, and its nucleotide sequence was determined. The cDNA clone encodes a polypeptide with a deduced amino acid sequence of 1145 residues and a predicted molecular mass of 129.9 kDa. Amino-terminal sequence analysis of the mature catalytic subunit of the heterodimeric mitochondrial enzyme from Drosophila embryos identified the amino-terminal amino acid at position +10 in the deduced amino acid sequence, indicating a mitochondrial presequence peptide of only nine amino acids. Alignment of the catalytic subunit sequence with that of Escherichia coli DNA polymerase I Klenow fragment indicated a high degree of amino acid sequence conservation in each of the three DNA polymerase and three 3' --> 5' exonuclease domains identified by biochemical studies in the latter enzyme. Bacterial overexpression, purification, and biochemical analysis demonstrated both 5' --> 3' DNA polymerase and 3' --> 5' exonuclease in the recombinant polypeptide. This represents the first demonstration of 3' --> 5' exonuclease activity in the polymerase catalytic subunit of animal mitochondrial DNA polymerase.