Drosophila mitochondrial DNA polymerase, pol gamma, is a heterodimeric complex of catalytic subunit and accessory subunits. Physical interactions between the two subunits were investigated by deletion mutagenesis in both in vivo reconstitution and in vitro protein overlay analyses. Our results suggest that the accessory subunit may consist of three domains, designated the N, M, and C domains. The M and C regions comprise the major contacts involved in subunit interaction, likely with multiple sites in the exonuclease (exo) region and part of the spacer between the exo and DNA polymerase (pol) regions in the catalytic subunit. Furthermore, the N region in the accessory subunit may modulate subunit assembly and/or conformation through weak interaction with the pol region in the catalytic subunit. Sequence comparisons identify a significant similarity between the M region of the accessory subunit and the RNase H domain of HIV-1 reverse transcriptase. On the basis of these results, the proposed function of the C-terminus of the accessory subunit in RNA primer recognition, and previous observations that mitochondrial DNA polymerase is itself a reverse transcriptase, we propose that the overall conformation and arrangement of functional regions in the Drosophila pol gamma complex resemble those of HIV-1 reverse transcriptase.