The mitochondrial DNA polymerase from embryos of Drosophila melanogaster has been examined with regard to template-primer utilization, processivity, and fidelity of nucleotide polymerization. The enzyme replicates predominantly single-stranded and double-stranded DNAs: the rate of DNA synthesis is greatest on the gapped homopolymeric template poly(dA).oligo(dT), while the highest substrate specificity is observed on single-stranded DNA templates of natural DNA sequence. Kinetic experiments and direct physical analysis of DNA synthetic products indicate that the Drosophila DNA polymerase gamma polymerizes nucleotides by a quasi-processive mechanism. The mitochondrial enzyme demonstrates a high degree of accuracy in nucleotide incorporation which is nearly identical with that of the replicative DNA polymerase alpha from Drosophila embryos. Thus, the catalytic properties of the near-homogeneous Drosophila DNA polymerase gamma are consistent with the in vivo requirements for mitochondrial DNA synthesis as described in a variety of animal systems.