Mitochondrial DNA polymerase from Drosophila embryos has been characterized with regard to its mechanism of DNA synthesis in the presence of single-stranded DNA-binding protein from Escherichia coli. The rate of DNA synthesis by DNA polymerase gamma was increased nearly 40-fold upon addition of single-stranded DNA-binding protein. Processivity of mitochondrial DNA polymerase was increased approximately 2-fold, while its intrinsic rate of nucleotide polymerization was unaffected. Primer extension analysis showed that the rate of initiation of DNA strand synthesis by DNA polymerase gamma was increased 25-fold in the presence of single-stranded DNA-binding protein. Our results indicate that the stimulation of Drosophila DNA polymerase gamma by single-stranded DNA-binding protein results primarily from an increased rate of primer recognition and binding. Concurrent achievement of maximal activity and processivity by mitochondrial DNA polymerase in the presence of binding protein suggests that DNA polymerase gamma, like other replicative DNA polymerases, associates with accessory factors in vivo to catalyze efficient and processive DNA synthesis.