Mitochondrial protein extracts from normal and immortalized mammalian somatic cells catalyze homologous recombination of plasmid DNA substrates. Mitochondrial homologous recombination activity required exogenous adenosine triphosphate, although substantial activity remained when non-hydrolyzable analogs were used instead. There was no requirement for added nucleoside triphosphates, and the reaction was not inhibited by dideoxyadenosine triphosphate or aphidicolin. The majority of recombinant plasmid molecules result from a conservative process, indicating that nuclease-mediated strand-annealing is not responsible for the mitochondrial homologous recombination activity. Affinity-purified anti-recA antibodies inhibited the reaction, suggesting that activity is dependent on a mammalian mitochondrial homolog of the bacterial strand-transferase protein. The presence of homologous recombination activity within mammalian mitochondrial extracts suggests that this process is involved in mitochondrial DNA repair.