Aconitase, the second enzyme of the tricarboxylic acid cycle encoded byACO1in the budding yeastSaccharomyces cerevisiae, catalyzes the conversion of citrate to isocitrate.aco1Δ results in mitochondrial DNA (mtDNA) instability. It has been proposed that Aco1 binds to mtDNA and mediates its maintenance. Here we propose an alternative mechanism to account for mtDNA loss inaco1Δ mutant cells. We found thataco1Δ activated the RTG pathway, resulting in increased expression of genes encoding citrate synthase. By deletingRTG1,RTG3, or genes encoding citrate synthase, mtDNA instability was prevented inaco1Δ mutant cells. Increased activity of citrate synthase leads to iron accumulation in the mitochondria. Mutations inMRS3andMRS4, encoding two mitochondrial iron transporters, also prevented mtDNA loss due toaco1Δ. Mitochondria are the main source of superoxide radicals, which are converted to H2O2through two superoxide dismutases, Sod1 and Sod2. H2O2in turn reacts with Fe2+to generate very active hydroxyl radicals. We found that loss of Sod1, but not Sod2, prevents mtDNA loss inaco1Δ mutant cells. We propose that mtDNA loss inaco1Δ mutant cells is caused by the activation of the RTG pathway and subsequent iron citrate accumulation and toxicity.