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Mitochondria contain an independently maintained genome that encodes several proteins required for cellular respiration. Deletions in the mitochondrial genome have been identified that cause several maternally inherited diseases and are associated with certain cancers and neurological disorders. The majority of these deletions in human cells are flanked by short, repetitive sequences, suggesting that these deletions may result from recombination events. Our current understanding of the maintenance and repair of mtDNA is quite limited compared to our understanding of similar events in the nucleus. Many nuclear DNA repair proteins are now known to also localize to mitochondria, but their function and the mechanism of their action remain largely unknown. This study investigated the contribution of the nuclear double-strand break repair (DSBR) proteins Rad51p, Rad52p and Rad59p in mtDNA repair. We have determined that both Rad51p and Rad59p are localized to the matrix of the mitochondria and that Rad51p binds directly to mitochondrial DNA. In addition, a mitochondrially-targeted restriction endonuclease (mtLS-KpnI) was used to produce a unique double-strand break (DSB) in the mitochondrial genome, which allowed direct analysis of DSB repair in vivo in Saccharomyces cerevisiae. We find that loss of these three proteins significantly decreases the rate of spontaneous deletion events and the loss of Rad51p and Rad59p impairs the repair of induced mtDNA DSBs., Author Summary Mitochondria are the powerhouse of the cell, providing energy in the form of ATP. Several proteins required for the production of ATP are encoded in the mitochondrial genome, which is maintained independently from the nuclear genome. Mutations in mitochondrial DNA are responsible for several inherited diseases and are associated with certain cancers, neurological disorders, and aging. In human mtDNA, 90% of deletions, a specific type of mutation, are flanked by repetitive sequences. Deletions in this context are generally produced through homologous recombination, specifically single-strand annealing. For the first time, in this study we show that the proteins involved in nuclear homologous recombination, Rad51p, Rad52p, and Rad59p, also contribute to the formation of mitochondrial deletions. Our data also suggest a novel mitochondrial-specific role for Rad51p in the generation of this type of deletion. Further study of these repair pathways allows us to garner a better understanding of these processes that are involved in disease pathologies and aging.