Key components of the ascorbate-glutathione cycle in Arabidopsis cell organelles are encoded by single organellar targeted isoforms that are dual localized in the chloroplast stroma and the mitochondrion. We demonstrate the presence of the ascorbate-glutathione cycle in purified Arabidopsis mitochondria using enzymatic activity, proteomic and in vitro and in vivo subcellular targeting data that identify the gene products responsible. In vitro experiments using a dual import assay assessing mitochondrial and chloroplast imports simultaneously show dual targeting of ascorbate peroxidase, monodehydroascorbate reductase, and glutathione reductase gene products to mitochondria and chloroplasts, while a putative dehydroascorbate reductase protein is only imported into mitochondria. In vivo subcellular localization using green fluorescent protein fusion proteins show clear targeting of all gene products to mitochondria. Transcript levels show these genes are induced by oxidative chemical stresses targeted to chloroplasts and/or mitochondria and are elevated during photosynthetic operation in the light. Together these data present a model of an integrated ascorbate-glutathione antioxidant defense common to plastids and mitochondria that is linked at the level of the genome in Arabidopsis.