Site-specific gene manipulation using homologous recombination induced by a double-strand break in DNA is expected to be applied to mitochondrial genome manipulation. In this study, by using a chemistry-based DNA cutter, a double-strand break was formed in mitochondrial DNA. In highly purified mitochondrial extract, the scission mitochondrial DNA enhanced homologous recombination, confirming eminent activity of mitochondrial components for this important bioreaction.